Conformational and topological protein regulation

ABSTRACT

Methods and compositions are provided for identifying novel conformers of proteins not known to exist as different conformers. By using chimeric genes replacing a native signal sequence with a different signal sequence resulting in the production of a conformer, one can compare the native protein with the product of the chimeric gene. Where the conformations are different, the different protein may be used for production of antibodies, elucidation of mechanisms associated with the native and different conformer protein, assays for the presence of the different conformer in physiological samples, identification of compounds specifically binding to the conformer, particularly drugs, etc. Where the formation of the conformer is associated with a diseased state, the conformer may be used in screens to identify compounds as drug candidates.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This specification claims the benefit of U.S. Ser. No. 60/171,012filed Dec. 15, 1999 and U.S. Ser. No. 60/172,350 filed Dec. 16, 1999,which disclosures are incorporated herein by reference.

INTRODUCTION TECHNICAL FIELD

[0002] The field of this invention is biological models andtherapeutics.

BACKGROUND

[0003] Conventionally, gene expression in higher eukaryotes is generallybelieved to involve the conversion of information encoded in the genomeinto proteins through the processes of transcription, RNA splicing andexport, and translation. Localization of newly synthesized proteins tothe correct intracellular compartment, and folding into the correcttertiary structure, are two additional processes at which geneexpression could, in principle, be regulated. However, until now theformer has been viewed as regulated only with respect to fidelity (Songet al, Cell (2000)100:333-343), degradation (Wiertz et al, Nature (1996)384:432-438), or signaling to other compartments (Sidrauski et al.,Trends Cell Biol (1998) 8:245-249), and not with respect to theinformation content of the expressed gene. Likewise, folding has alsonot been viewed at a point of fundamental regulation of gene expression.In general, a dichotomy has been accepted between a protein being eitherproperly folded or misfolded (Ellgaard, et al., Science (1999)286:1882-1888). The possibility that proteins might have more than oneproperly folded state and that the cell might be able to select oneversus another folded state under particular circumstances orconditions, has not been seriously considered. If either of thesepossibilities were true, the information content of the genome would begreatly increased. If an invention made it possible to select oneconformation versus another, such an invention would be a platform fordetermining and accessing the information content of the genome in waysthat have not been heretofore possible.

[0004] In regards to protein folding, a fundamental dogma of modernbiology is that primary structure determines secondary structure, which,together with relevant post-translational modifications such asglycosylation, determines the tertiary structure of proteins (Anfinsen,Science (1973) 181:223-230). One revision of this view occurred with therealization that molecular chaperones play a crucial role in enhancingthe fidelity of protein folding by preventing inappropriateinteractions, thereby facilitating the process of achieving the properfinal folded state (Ellis and Hartl, Faseb J (1996) 10:20-26). Therecognition that folding is likely initiated in many parts of themolecule at the same time, allowing the chain to funnel towards aminimum energy state without sampling every possibility along the way,constituted a second revision in the generally accepted view of proteinfolding (Dill and Chan, Nature Struct Biol (1997) 4: 10). Neither ofthese notions considers the possibility that folding might be regulatedin the sense of proceeding down one versus another pathway contingent onone versus another set of protein-protein interactions. If this were thecase, protein folding could be amenable to manipulation in ways thatcould confer diagnostic or therapeutic advantage.

[0005] Proteins destined to be secreted from the cell generally containa signal sequence at the amino terminus that initiates a series ofprotein-protein interactions directing the growing chain to theendoplasmic reticulum (ER) membrane and through the translocationchannel into the ER lumen (Blobel, PNAS (1980) 77:1496-1500). The rolesof signal recognition particle and its receptor (Walter and Johnson,1996) and of the heterotrimeric Sec 61 complex (Gorlich and Rapoport,Cell (1993) 75:615-630) and of other proteins (Jungnickel and Rapoport,Cell (1996) 82:261-270) in these processes has been elucidated.

[0006] Assembly of integral membrane proteins into the membrane of theER appears to be a complex variation on this general theme, directed byinternal signal sequences and stop transfer sequences and hybridsignal-anchor sequences whose interaction with various ER proteinsdirects the final transmembrane orientation of the polypeptide and oftenplay a subsequent role in anchoring the protein in the bilayer after thechain is released from the translocation channel into the lipid bilayer(Skach and Lingappa, J Biol Chem (1993) 268:23552-23561; Borel andSimon, Cell (1996) 85:379-389; Heinrich et al, Cell (2000) 102:233-244;Moss et al., Mol Biol Cell (1998) 9:2681-2697).

[0007] In the case of secretory proteins, the signal sequence is usuallycleaved from the growing chain by the ER membrane associated signalpeptidase complex, trapping the nascent chain in the ER lumen (Matlacket al., J Cell Biol (1999) 270:6170-6180), with the cleaved signalpeptide most likely returned to the cytosol and degraded (Lyko et al., JBiol Chem (1995) 270:19873-19878). In bacteria and primitive eukaryotessuch as yeast, a substantial amount of translocation occurs aftersynthesis is completed (Rapoport et al., J Biol Chem (1999)380:1143-1150). In such post-translational translocation, a role for thesignal sequence as a molecular chaperone to maintain the unfolded statehas been proposed (Liu et al., PNAS (1986) 86:9213-9217). However inhigher eukaryotes, where most translocation across the ER membraneappears to occur co-translationally, that is, while the chain is stillbeing made, it has generally been assumed that the nascent chain istransferred directly to the ER lumen where folding is initiated (Chenand Helenius, Mol Biol Cell (2000) 11:765-772).

[0008] Taken together, the studies presented here suggest the need forseveral revisions in the current paradigm of protein folding.

[0009] First, the simple dichotomy between properly folded and misfoldedproteins must be abandoned. It needs to be recognized that proteinfolding can result in multiple properly folded states which may,potentially, subserve different functions. In most cases these areextremely difficult to detect not only because tools to easilydistinguish conformational variants of proteins are limited and noteasily applied, but also because the cell complicates the task bydegrading variants not wanted at a given point in time.

[0010] Second, it must be recognized that the cell has mechanisms bywhich one folded state (and therefore one function) is chosen (tosurvive degradative mechanism and be exported to the surface or out ofthe cell at one time while another folded state (and another function)may be chosen at another time.

[0011] Third, it is clear that the machinery and determinants involvedin translocation across the ER membrane play an important role inselecting the folding funnel down which a newly synthesized proteinproceeds, and that manipulation of either the signal sequence or themachinery with which the chain interacts in the cytosol, membrane or ERlumen are ways to change the folding funnel selected, and therefore, thefinal conformation or mix of conformations of the protein.

[0012] The major implication of the first two revisions of the proteinfolding paradigm is to increase the information content of the genomeenormously. The major implication of the third is to reveal a means ofaccessing this increased information content of the genome fordiagnostic and therapeutic advantage, giving rise to the subjectinvention.

[0013] Besides elucidating these points of background, our studiesdemonstrate an invention by which the choice of folding funnel can bealtered for any given protein in a way that does not change the proteinsfinal primary structure, but which can result in dramatic changes in theprotein's final folded conformation or shape. We demonstrate the signalsequence is able to influence the folding funnel utilized by the growingpolypeptide chain, and thereby, to influence the final foldedconformation of the resulting newly synthesized protein. Furthermore,cells are able to choose one versus another final conformation throughsignal sequence-mediated regulation. Because a linear polypeptidesequence folding in different ways could have substantially differentshapes, physical properties and biological activities, this new level ofregulation greatly increases the information content of the genome andthe potential for regulation of gene expression available to the cell.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1 shows the effects on topology of replacing the PrP signalsequence. FIG. 1(A) shows schematic diagrams of wild-type PrP, Prl-PrP,and βL-PrP. Boxes represent signal sequences from PrP, preprolactin, andpre-beta-lactamase, fused to the PrP mature region. Cleavage site isindicated by arrowhead. FIG. 2(B) shows the topology of PrP, Prl-PrP,and βL-PrP. Microsomes from in vitro translation and translocationreactions of each construct were isolated by sedimentation and dividedinto equal aliquots for digestion with proteinase K (PK) in the presenceor absence of 1% Triton X-100 (detergent) as indicated below the gel.One aliquot was left untreated. The three topological forms of PrP yielddistinctively sized bands upon digestion with PK in the absence ofdetergent (Hedge et al. (1988a) Science 279, 827-834). The position ofeach band, along with a diagrammatic representation of the respectivetopology, is provided to the left of the gel. Cleavage of the signalsequence of Prl-PrP was inefficient, causing ^(sec)PrP and ^(Ntm)PrP tobe represented by both a signal-cleaved species and a signal-uncleavedspecies. Molecular weight markers are at right. The percentage oftranslocated chains in the ^(Ctm)PrP topology for each construct wasquantitated from 3 such experiments and is shown in (C) (mean+/−SEM).

[0015]FIG. 2 shows that kinetics of targeting do not influence PrPtopology. FIG. 2 (A) shows PrP translated in the presence of varyingamounts of microsomes as indicated on the x-axis. Following proteolysisassays for topology, autoradiographs were quantitated to determine thepercentage of total translation product that was signal cleaved (closedsquares), and the percentage of translocated chains in the ^(Ctm)PrPtopology (open circles). FIG. 2(B) shows translation reactions of PrPsynchronized by inhibition of initiation at 5 minutes with 75 mM ATA. Atstaggered intervals (as indicated on the x-axis), aliquots were removed,microsomes were added, and targeting and translocation were allowed toproceed for 30 minutes. Topology and signal cleavage were determined andplotted as in FIG. 2(A). FIG. 2(C) shows an StuI-truncated mRNA,encoding the 223-mer of PrP, translated in the presence (open bars) orabsence (shaded bars) of microsomes. After 5 minutes, further initiationwas inhibited with ATA, and at 30 minutes, microsomes were added to thesample lacking them and the incubation for both reactions was continuedfor an additional 30 minutes. Nascent chains were then released from theribosomes with 10 mM EDTA. Signal cleavage and topology were assessedand quantitated as above.

[0016]FIG. 3 shows that the signal sequence influences theribosome-membrane junction. FIG. 3(A): Truncated mRNAs for preprolactin(Prl), pre-beta-lactamase (βL), βL-Prl and Prl-βL were translated andtranslocated, and the microsomes from these reactions were isolated bysedimentation. One aliquot of each sample was left untreated and PK wasadded to the remainder. Prl and βL-Prl were truncated at PvuII. βL andPrl-βL were truncated at HpaII. The sizes of these constructs in aminoacids are indicated. FIG. 3(B): Prl-PrP, wild-type PrP, and βL-PrP weretruncated at BstXI (61 aa for wild-type PrP), NaeI (113 aa), XbaI (135aa), or HincII (180 aa) and analyzed by proteolysis as in FIG. 3(A). Thepercentage of material protected from digestion by PK was measured bydensitormetric quantitation of the autoradiographs shown, and is givenbelow each sample. The drawings at right represent the position of boththe signal sequence (open box) and transmembrane domain (gray box) withrespect to the ribosome.

[0017]FIG. 4 shows dissociation of topology from TRAM dependence. FIG.4(A): signal sequences of constructs PrP, PrP_((R2, 3)), PrP_((D2, 3)),PrP_((R4, 5)), and PrP_((D4, 5)). The amino acid changes in eachconstruct are underlined, and the site of signal cleavage is indicatedby an arrowhead. FIG. 4(B): the constructs shown in FIG. 4(A) were usedto program translation and translocation reactions, and the resultanttopology analyzed by proteolysis as in FIG. 1B. The percent of ^(Ctm)PrPsynthesis was quantitated by densitometry of the autoradiographs and isshown for each construct. The signal sequence of each construct isindicated on the x-axis. ^(Ctm)PrP synthesis for Prl-PrP and βL-PrP isshown for comparison. FIG. 4(C): the PrP signal sequence mutations shownin FIG. 5A were fused to the prolactin mature region. The signalsequences of prepro-alpha factor (dependent) and preprolactin(independent), fused to the prolactin mature region, served as controlsfor TRAM-dependent and TRAM-independent signal sequences, respectively(see Voigt et al., (1996) J. Cell. Biol. 134, 25-35). Constructs weretranslated and translocated in ConA-depleted proteoliposomes containingor lacking purified TRAM (prepared as described in the ExperimentalProcedures). An aliquot from each reaction was set aside to verify thatthe efficiency of translation was identical in both types ofproteoliposomes. The remainder of the material was treated with 0.5mg/ml PK and immunoprecipitated with anti-protein antibodies. TRAMdependence, shown here, was measured as the percent diminishment ofprotease protected material in the proteoliposomes lacking TRAM relativeto TRAM-containing membranes.

[0018]FIG. 5 shows relationship between the signal sequence and TMdomain. FIG. 5 (A): Constructs PrP_((AV3)), Prl-PrP_((AV3)), andβL-PrP_((AV3)) were translated, translocated, and analyzed for topologyby proteolysis exactly as in FIG. 1B. The migration of each topologicalform is indicated at right. FIG. 5(B): Constructs PrP_((G123P)),Prl-PrP_((G123P)), and βL-PrP(G123P) were translated, translocated, andanalyzed for topology by PK exactly as in FIG. 1B. Only ^(sec)PrP isobserved, and its migration is indicated at right. FIG. 5 (C):HincII-truncated mRNAs coding for ˜180-mers of PrP, Prl-PrP_((AV3)), andβL-PrP_((G123P)) were translated in the presence of microsomes, whichwere then isolated by sedimentation. Proteolysis with PK to examine thestate of the ribosome-membrane junction was performed exactly as in FIG.3B.

[0019]FIG. 6 shows the interaction of ^(SBC)PrP with PDI. FIG. 6(A):HincII-truncated mRNAs coding for PrP_((G123P)) and PrP_((AV3))(˜180-mers) were translated, and the membranes were isolated bysedimentation. One aliquot was treated with 1 mM DSS (+XL) and theremainder was left untreated (−XL). Uncrosslinked material is shown atthe bottom of the gel. The migrations of the molecular weight markersare shown at right. The arrowhead indicates a protein of approximately65 kDa which cross-links preferentially to PrP_((G123P)). FIG. 6(B):Prl-PrP and βL-PrP were truncated at HincII and treated exactly as inFIG. 6(A). Equal amounts of total translation product were loaded ineach lane. The 65 kDa cross-link seen in (A) is indicated by anarrowhead. FIG. 6(C): Prl-PrP and βL-PrP were truncated at NaeI(˜113-mers) and analyzed by cross-linking exactly as in FIG. (A). The 65kDa cross-link seen in FIG. 6(A) (closed arrowhead) and a 60 kDacross-link which also preferentially forms with Prl-PrP (open arrowhead)are indicated. FIG. 6(D): Purification of PDIp. Shown is a Coomassiestain of starting rough microsomes (RMs); proteins not extracted(pellet) or extracted by saponin treatment; flow-through (FT) and eluateof the saponin extract from a ConA-sepharose column; flow-through andeluate of the ConA eluate from a Q-sepharose column, and the pooled peakfractions from a gel-filtration (GF) column, resulting in purificationof p65 (arrow) to near-homogeneity. See Experimental Procedures fordetails of the purification. FIG. 6(E): Prl-PrP and β-PrP weretranslated in the presence of either pancreas (top panel) or brain(bottom panel) microsomes, and the membranes were isolated bysedimentation. Following cross-linking with 1 mM DSS, the nascent chainswere released from the ribosomes with 10 mM EDTA, and 0.5% saponin wasadded to extract the lumenal proteins. One aliquot was set aside (lanes1, 4), and antibodies against PDIp (lanes 2, 5) or PDI (lanes 3, 6) wereadded directly to the remaining material for immunoprecipitation.Prl-PrP translated in pancreatic microsomes cross-links preferentiallyto PDIp (closed arrowhead) and PDI (open arrowhead), while only across-link to PDI (open arrowhead) is observed in brain microsomes. FIG.6(F): mRNAs encoding wild-type PrP and signal sequence point mutantsfrom FIG. 4A, truncated at NaeI to yield ˜-113-mers, as well aswild-type preprolactin (Prl) truncated at MboII to yield a 100-mer, weretranslated and analyzed by cross-linking as in panel (A). Followingcross-linking and nascent chain release with 10 mM EDTA, 0.5% saponinwas added, the membranes were sedimented, and the supernatant,containing lumenal proteins, was recovered. Arrowheads designate^(sec)PrP-specific lumenal cross-links. The signal sequence of eachconstruct is indicated below the gel.

[0020]FIG. 7 shows a hypothetical model for signal sequence action. Thesignal sequence determines the state of the ribosome-membrane junction,which in turn influences final topological fate in the case of PrP, andperhaps more subtle aspects of translocational behavior or othersubstrates. An example of each translocational outcome is given.

[0021]FIG. 8 shows a dose response of ^(Ctm)PrP inducedneurodegeneration. FIG. 8(A): Topology of wild type and mutant PrPmolecules at the ER. In vitro synthesized transcript coding for each PrPconstruct (indicated above the gels) was used to program a rabbitreticulocyte lysate cell-free translation reaction containing ER derivedmicrosomal membranes and a competitive peptide inhibitor ofglycosylation. Following translation, samples were either left untreatedor digested with PK in the absence or presence of 0.5% Triton X-100(“Det”) as indicated above the gel. The positions of the full-length PrPmolecule, the NH₂-terminal fragment (indicative of ^(Ntm)PrP) and theCOOH-terminal fragment (indicative of ^(Ctm)PrP) generated by PKdigestion are marked at the right. FIG. 8 (B): Level of expression ofvarious transgenic mouse lines. Varying amounts in (μl, indicated abovethe lanes) of 10% brain homogenate from each transgenic mouse wasimmunoblotted for PrP with 13A5 monoclonal antibody and compared to atitration of syrian hamster brain homogenate. FIG. 8(C): Time course ofdevelopment of symptoms in Tg[SIIaPrP(A117V)_(H)] (closed circles) andTg[SHaPrP(N108I)_(H)] (open circles). Non-transgenic control animals(dashed line) and Tg[SHaPrP(KH→II)_(H)] (closed squares; data fromManson, et al. (1994) Neurology 46:532-537) are shown for comparison.FIG. 8(D): Analysis of various transgenic mice and Syrian hamster (asindicated above the gel) for ^(Ctm)PrP and Prp^(Sc) as described inMethods. Tg[SHaPrP(A117V)_(H)] and Tg[SHaPrP(N108I)_(H)] samples werefrom clinically ill mice and Tg[SHaPrP(A117V)_(L)] andTg[SHaPrP(N108I)_(L)] samples were from mice (which showed no signs ofdisease) at least 600 days of age. The fragment of PrP resistant to PKunder the ‘mild’ conditions, indicative of ^(Ctm)PrP is only seen in theTg[SHaPrP(A117V)_(H)] and Tg[SHaPrP(N108I)_(H)] samples. No PK resistantPrP^(Sc) was observed in any of the samples.

[0022]FIG. 9 shows a relationship between ^(Ctm)PrP and PrP^(Sc). FIG.9(A), (C), (E): Time course of development of illness in varioustransgenic lines following inoculation with Sc237 hamster prions. FIG.9(B), (D), (F): Relative levels of protease-resistant Prp^(Sc) at timeof illness in various transgenic lines. Duplicate samples of each linewere digested using ‘harsh PK’ conditions as described in Methods andequivalent amounts of each sample separated by SDS-PAGE. The C-terminalPrP27-30 fragment characteristic of PrP^(Sc) (indicated with a bracket)was detected by immunoblotting with the 13A5 monoclonal antibody. Theposition of undigested, full length PrP is indicated with an asterisk.FIG. 9(G): The Ctm-index for each transgenic line (see Table 1) wasplotted against the amount of PrP^(Sc) accumulated at the time ofillness following inoculation with Sc237 prions.

[0023]FIG. 10 shows a lack of transmission of Ctm PrP inducedneurodegenerative disease. Terminally ill Tg[SHaPrP(KH→II))_(H)] mice(‘KH→II’) and clinically normal Tg[SHaPrP] mice (‘wt’) were sacrificedand homogenates of the brain tissue inoculated intracerebrally intovarious hosts as indicated below the graph. The host animals used wereTg[SHaPrP(KH→II)_(L)] [‘(KH→II)_(L)’], FVB/Prnp^(0/0) (‘PrP null’),Tg[SHaPrP], and Syrian hamsters. The time (in days) at which individualanimals died following inoculation is plotted. Deaths by all causes,including those related to the inoculation itself, are plotted. Theexperiments were terminated after ˜600 days with none of the remaininganimals showing any signs or symptoms of neurologic disease. Eachexperiment represents three sets of 10 host animals injected withinoculi prepared from three separate animals to be tested. It should benoted that in our animal care facility, hamsters routinely liveapproximately 200 to 400 days, while mice last greater than 600 days(data not shown).

[0024]FIG. 11 shows ^(Ctm)PrP generation during time course of PrP^(Sc)accumulation. FIG. 11(A): Schematic of experimental design. Doubletransgenic mice expressing both SHaPrP and MoPrP (represented by theshaded and open circles, respectively) are inoculated with RML mouseprions (crosshatched squares). Over time, host MoPrP^(C) is converted toMoPrP^(Sc) and accumulates. During this time course, the HaPrP is notconverted to HaPrP^(Sc) owing to the species barrier and may thereforebe assayed for ^(Ctm)PrP. FIG. 11(B): Relative amounts of total PrP^(Sc)and SHaPrP^(Sc) in mice at various times (in weeks) after inoculationwith RML. Homogenate was digested using the ‘harsh PK’ conditions,treated with PNGase, and analyzed by SDS-PAGE and immunoblotting witheither R073 polyclonal antibody (to detect total PrP) or the 3F4monoclonal antibody (to selectively probe for SHaPrP). An equivalentamount of homogenate is analyzed in each lane except lanes 2 and 11(which contain one-fourth as much) and lanes 3 and 10 (which containone-tenth as much). FIG. 11(C): Relative amounts of hamster ^(Ctm)PrP(detected selectively using the 3F4 monoclonal antibody) at varioustimes after inoculation with RML. Each bar represents the average ±SEMof three determinations.

[0025]FIG. 12 shows a three stage model of prion disease pathogenesis.Stage I is the formation and accumulation of PrP^(Sc). This could beinitiated by either inoculation or spontaneous conversion of a mutatedPrP^(C) to PrP^(Sc). Stage II comprises the events involved ingenerating ^(Ctm)PrP. These events could be affected in trans at apresently unknown step (dashed lines with question marks) by accumulatedPrP^(Sc) or in cis by certain mutations within PrP. Stage III representsthe events (currently unknown) involved in ^(Ctm)PrP mediatedneurodegeneration. This likely involves exit of ^(Ctm)PrP to a post-ERcompartment as a first step. In this model, PrP^(Sc) can cause disease(via ^(Ctm)PrP), but is not absolutely necessary, whereas ^(Ctm)PrPproduction is necessary and sufficient for the development of disease.

[0026]FIG. 13 shows efficiency of model secretory protein signalsequences. Full-length mRNAs for preprolactin (Prl), pre-beta-lactamase(βlac), and pre-IgG heavy chain (IgG) were translated in a rabbitreticulocyte lysate in the absence or presence of canine pancreaticmicrosomal membranes (RM), and equal aliquots of the translated materialwere left untreated or treated with Proteinase K (PK) in the presence orabsence of 1% Triton X-100 (det). The positions of unprocessed material(pPrL, pβlac, plgG) are indicated, as are the positions ofsignal-cleaved (Prl, βlac) and glycosylated (IgG-CHO) material.

[0027]FIG. 14 shows that nascent secretory proteins act differentiallyon the ribosome-membrane junction. FIG. 14(A): Preprolactin MRNA wastruncated at successive locations giving rise to nascent chainscontaining the noted number of N-terminal amino acids. The number ofamino acids of the signal-cleaved protein is given in parentheses. Thestate of the ribosome-membrane junction was assessed by proteolysisexactly as in Rutkowski et al. (“A New Role for the Signal Sequence inTranslocational Regulation”, see priority applications 60/171,012 and60/172,350) FIG. 14(B) and (C): βlac and IgG mRNAs were seriallytruncated at the indicated locations and analyzed by proteolysis as in(A). For each panel the cartoon to the right depicts the state of theribosome-membrane junction at each of the three points during chaingrowth. Note that the left panel only of FIG. 14A and the left andcenter panels of B and C depict an “open” ribosome-membrane junction,while the center and right hand panels of FIG. 14A and the right handpanels of FIG. 14B and C depict a “closed” ribosome-membrane junction.

[0028]FIG. 15: Prolactin (Prl) and IgG/Prl were truncated at PvuII(encoding the signal sequence and 56 amino acids of the mature region ofeach protein) and translated in the presences of canine pancreatic roughmicrosomes. Targeted chains were isolated by sedimentation. One aliquotwas set aside and the other was treated with 2 mM BM(PEO)3 [XL]. Thearrowhead indicates a protein of approximately 35 kDa which cross-linkspreferentially to Prl. 10 kDa uncross-linked material is at the bottomof the panel. Migration of molecular weight markers is indicated to theleft of the gel.

[0029]FIG. 16: Glycosylation as a reporter of signal sequence-dependentconformational change. FIG. 16(A): in vitro-transcribed mRNAs encodingnative preprolactin (Prl) with an engineered N-terminal glycosylationsite, or glycoprolactin fused to the signal sequences of mouse IgG heavychain (IgG/Prl), rat growth hormone (GH/Prl), or hamster PrP (PrP/Prl),were translated in a rabbit reticulocyte lysate system, either in thepresence or absence of canine rough microsomes (RM) as indicated. Acompetitive inhibitor of glycosylation was also added to some samples(AP). Following translation, samples were either set aside or incubatedwith 0.5 mg/ml proteinase K (PK) in the presence or absence of 1% TritonX-100 (det) at 0° for 30 minutes. The reactions were then run onSDS-PAGE and visualized by autoradiography. Three species of prolactinare indicated: signal-cleaved, translocated, glycosylated prolactin(Prl-CHO); signal-uncleaved untranslocated preprolactin (pPrl); andsignal cleaved, translocated, nonglycosylated prolactin (Prl). FIG.16(B): The percentage of prolactin chains achieving glycosylation foreach construct was quantitated from three experiments by scanning anddensitometric analysis of the autoradiographs such as those shown in(FIG. 16(A). FIG. 16(C): COS-1 cells were transiently transfected withexpression plasmids encoding glycoprolactin with its own signal sequenceor those of rat growth hormone or mouse IgG heavy chain. For comparison,the same constructs lacking glycosylation sites were also transfected(—CHO). Following a 45 minute preincubation of the cells in serum-freemethionine-free medium, 100 μCi of a ³⁵S-Methione/Cysteine mixture wasadded to the cell medium and the cells were labeled for 1 hour. Analiquot of the media from these cells was run directly on SDS-PAGE. Bothglycosylated and unglycosylated forms of prolactin are indicated to theright of the panel. While glycosylated GH/Prl and IgG/Prl are clearlyvisible, no glycosylated native prolactin is observed.

[0030]FIG. 17 shows requirement of junctional regulation for successfultranslocation. FIG. 17(A): The prolactin mature region was fused toeither its own signal sequence (Prl-Prl) or those of pre-beta-lactamase(β-Prl) or pre-IgG heavy chain (IgG-Prl) at the site of signal sequencecleavage. Full-length mRNAs for these substrates were translated in thepresence of microsomal membranes and analyzed as in FIG. 17A. For eachsubstrate, the efficiency of translocation was determined byquantitating the percentage of total chains which achieved a processed,translocated state. Each bar represents the mean percent translocationfrom three trials (+/−SEM). FIG. 17(B): mRNAs encoding Prl-Prl, βL-Prl,and IgG-Prl were truncated at either PvuII or AflII. The number of aminoacids from mature prolactin is shown in parentheses. Translated,sedimented chains were analyzed by proteolysis as in FIG. 15B to assessthe state of the ribosome-membrane junction. The y-axis plots the meanpercent protection of each substrate from PK in three trials. FIG.17(C): Schematic of IgG-Prl chimeras. Increasing lengths of pre-IgGheavy chain or a non-IgG stretch of amino acids were fused to theprolactin mature region. The site of signal sequence cleavage is shownby an arrowhead. Sequences from IgG are represented by open boxes, Prlmature region sequences by bars, and the non-IgG control sequence by ahatched box. The Prl signal sequence is indicated by a shaded box.Numbers below each construct represent amino acids from pre-IgG heavychain, the preprolactin signal sequence (Prl-Prl), or the non-IgGsequence (numbers 15-132 of IgG-Prl(1-132)Stuffer) with the initiationmethionine as number 1. FIG. 17(D): The substrates shown in FIG. 17(C)were translated in the presence of microsomal membranes and analyzed forthe mean percent translocation from three trials as in FIG. 17(B).

[0031]FIG. 18 shows a schematic of how translocational regulation canlead to conformational heterogeneity. Indicated in Roman numerals to theleft are the endpoints of three stages of protein biogenesis at the ER:I, the earliest events including targeting; II, the events oftranslocation per se; and III, the final folded protein. Translocationalregulation, of which four forms are indicated as FIG. 18(A-D) in stageII, provides the means by which heterogeneity is achieved amongcompleted, folded proteins (see III), as hypothesized here. Molecularchaperones are indicated by solid ovals, while TrAFs are depicted ashatched rectangles. In FIG. 18(A), the translocon serves as a molecularchaperone. In FIG. 18(B), the translocon forces the nascent chain toinitiate folding in a reducing environment, perhaps in association withmolecular chaperones or machinery for post-translational modifications.Note that the lumenal gate of the translocon is closed, while that onthe cytoplasmic side that makes up the ribosome-membrane junction, isopen. In FIG. 18(C), the converse is achieved—a closed cytoplasmic gateand an open lumenal gate, again with the implicit participation ofdistinct molecular chaperones. Finally, FIG. 18(D) indicates that theaction of TrAFs can result in a change in protein topology as well asconformation. First order protein folding is independent of theseconsiderations. Second order protein folding manifests itself indifferent ways (note different molecular chaperones in FIG. 18 B vs C)depending on TrAF action which constitutes the third order level ofcomplexity of protein folding. The final order of complexity of proteinfolding is due to the regulation of TraAFs by signaling pathways thatallow cells to choose between FIGS. (A-D), for example, in response to achange in environmental or other conditions.

[0032]FIG. 19: PrP cDNA was transcribed and translated as describedpreviously (Lopez et al, Science (1990 248:226-229) in the presence ofcytosolic extract prepared from mouse erythroleukemia cells before and 4of 6 days after induction of differentiation with dimethylsulfoxide.Thee presence of 35S methionine during the translation reactions allowsradiolabelled newly synthesized proteins to be visualized bypolyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE)and autoradiography (AR). One unit of activity is the amount of extractneeded to change the ratio of PrP topological forms in favor of sec PrPin a 10 μl translation reaction supplemented with dog pancreasmicrosomal membranes at a final concentration of 5 A280 u/ml. To preparethe cytosolic extracts, cells were dounce homogenized after swelling in10 mM Hepes pH 7.5 and membranes removed by centrifugation at 100,000×gfor 1 hr. As can be seen, cytosols from undifferentiated MEL cells arerich in an activity that promotes secPrP, and that activity is lost upondifferentiation of the cells, over time. In this case PrP is serving asa reporter of cytosolic regulatory activities that are likely utilizedby several proteins for different endpoints. Note the presence of aglobin band in the gel from the 6 day point, indicating induction ofglobin, a differentiation marker in these cells.

[0033]FIG. 20: Transcription and translation of PrP cDNA was performedas previously described in the presence of microsomal membranes from theindicated sources. The products were subjected to protease digestion inthe absence or presence of detergent as previously described. Shown arethe products of digestion with protease in the absence of detergent,allowing the ratio of Sec-PrP to Ctm-Prp to be readily assessed bySDS-PAGE and AR. E=microsomal membranes from hamster brain on embryonicday 13; N=neonatal hamster brain microsomal membranes; 7d=microsomalmembranes from 7 day post natal hamster brain; 14d=microsomal membranesfrom 14 day post natal hamster brain; 21d=microsomal membranes from 21day post natal hamster brain; A=microsomal membranes from adult hamsterbrain; SUE=sea urchin embryo microsomal membranes (which lack TrAF andtherefore generate exclusively Ctm-PrP comparable toglycoprotein-depleted fractionated and reconstituted proteoliposomes(Hegde et al., (1998) Mol Cell 2:85-91). Upper arrow indicates theposition of SecPrP; lower arrow of Ctm-PrP. Graph below isquantification of the ratio of secretory PrP to total PrP for each ofthe indicated lanes above. What is demonstrated is that membranes fromdifferent tissues and species, and from the same tissue and species atdifferent times in development, can have radically different TrAFactivities, consistent with the hypothesis that translocation is animportant site of regulation of protein biogenesis, and thattrans-acting factors contribute to translocational regulation. In thiscase, prion protein is being used solely as a reporter of TrAF activity.The same principle applies to all genes for which translocationalregulation may be found.

SUMMARY OF THE INVENTION

[0034] Methods and compositions are provided for producing proteins ofvarying topography and/or topological species, providing chimericproteins, identifying specific agents involved with the formation oftopographical and/or topological species, model in vitro and in vivosystems, and methods for identifying topographical and/or topologicaldistinct proteins. Proteins of varying conformation are produced byvarying the signal sequence, replacing the wild-type signal sequencewith sequences of known mechanism, selectively including transloconassociated proteins in an in vitro translocation model system, employingmodified lysates with microsomes for investigating and producingconformationally distinct proteins, for producing and employingknock-out and mutant small laboratory mammals resulting in modulation ofthe topographical production of target proteins and elucidatingphysiological mechanisms.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

[0035] Methods and compositions involving elements of the proteintranslocating system are employed in elucidating components of thesystem and their function, modulating folding of proteins using chimericgenes employing an unnatural signal sequence to provide “conformers,”(proteins having at least substantially the same amino acid sequence,but different physical topology or topography). By topology is intendedthe different placement of the protein, e.g. C-cytosolic as compared toN-cytosolic, and topography intends change in external conformation orshape, identifying conformers, providing in vitro and in vivo systemsfor these purposes, and identifying compositions that modulate proteinconformation during translocation. Topography as used herein refers toproteins of substantially the same or similar amino acid sequence butdifferent three dimensional shapes due to differences infolding/conformation. As used herein, polypeptides of substantially thesame amino acid sequence are those with conservative amino acidsubstitutions (i.e. a small or large side chain for a small or largeside chain, respectively; or an acidic, basic, polar or hydrophobic sidechain for an acidic, basic, polar or hydrophobic side chain,respectively), that do not alter the protein conformation or topology.Methods for identifying the multiple gene products that regulate signalsequence mediated selection of folding funnels during protein biogenesisalso are provided, using a reticulocyte lysate fractionation scheme. Themethodology for fractionation of translation extracts and fractionationof solubilized membranes with reconstitution of subfractions containingor missing particular trans-acting activities, offers several advantagesover existing systems. The process can be readily expanded to a largescale; the materials required for the fractionation are eitherinexpensive (common chemicals) or reuseable (centrifuge tubes, ionexchange resin); and the final products have a long shelf life whenstored appropriately.

[0036] This fractionated system offers several advantages over thecurrently available RRL, including: We have demonstrated that simplysupplementing an S-100 fraction from Xenopus oocytes with RRL ribosomesresults in translation efficiencies more than an order of magnitudegreater than previous oocyte translation systems (2). Such an oocytesystem can be used to study oocyte specific events such as translationalrepression of developmentally regulated factors; and also, it appearsthat the ribosomes contained in the rough microsome preparation from dogpancreas are highly active in restoring translation to the DEAEfraction. Thus, translocation events can be studied in an essentiallyhomologous system in which dog pancreas ribosomes are translating andtranslocating proteins across dog pancreas membranes. Subtle andregulated interactions between ribosomes and translocon proteins may bemore faithfully reproduced in a homologous system versus a heterologoussystem.

[0037] This system can be readily adapted to other translation systemssuch as the wheat germ translation system, or Xenopus oocyte translationsystem. In some cases, the fractionated system results in an enormousincrease in the translation efficiency (see above). Furthermore, theability to mix and match components from multiple such fractionatedsystems allows tissue specific events involved in the biogenesis ofcertain proteins to be studied. This is potentially useful for theidentification of factors, by complementation, involved in such tissuespecific events. Examples of such tissue specific differences have beendocumented (Wolin and Walter, J. Cell Biol (1989) 109:2617-2622; Lopezet al., Science (1990) 248:226-229).

[0038] The proteins of interest are proteins, which have a signalsequence and are subject to processing in the endoplasmic reticulum.Numerous signal sequences have been identified from different proteinsand appear to be capable of operating conjugated to a broad range ofunnatural proteins. The signal sequences are usually N-terminal, but maybe internal to the protein or C-terminal. Signal sequences are selectedfrom proteins that are known to have a specific mechanism fortranslocation affecting the conformation of the product or may besynthetic, where the translocational effect is known or determined. Itis now known that the signal sequence affects the conformation of theprotein that is translated in conjunction with the translocon. Withoutbeing bound by any theory, the signal sequence directs whether theribosome forms a tight, loose or intermediate junction with theendoplasmic reticulum (ER) and the selection of the channel andaccompanying processing proteins through which the translated protein istranslocated and processed. For example, replacing a signal sequence ofa protein with a signal sequence from preprolactin results in a tightjunction, while the signal sequence from pre-β-lactamase provides aloose junction. Proteins which provide tight junctions include:growthhormone; and loose junctions include: immunological heavy chain andyeast alpha-factor; and intermediate junctions include ductin,calreticulin, PrP, angiotensinogen and MDR-1, where the division betweenthe different conformers may be attributed to a variety of mechanisms.(See, for discussion, Hegde and Lingappa, (1996) Cell 85, 217-228 for adiscussion of the effect of the ribosome-membrane junction.) For ageneral discussion of the mechanism of translocation, see, for example,Ellgaard, et al., (1999) Science 286, 1882-1888; Wickner, et al., (1999)Science 286, 1888-1893; and Ibba and Soll, (1999) Science 286,1893-1897.

[0039] Signal sequences may be rated by using a lysate competent forexpression, microsomes and proteinase K. The degree of proteolysisoccurring with different signal sequences and a common gene isindicative of the nature of the junction of the ribosome with the ER. Byusing signal sequences having different degrees of junction tightness,the conformation of the resulting protein can be modified. Thesedifferent conformers may be used in a number of ways. The conformers maybe used for the production of antibodies, either antisera or preferablymonoclonal antibodies. The antisera and antibodies are prepared inconventional ways using the different protein conformers to immunize ahost, usually a mouse in the case of monoclonal antibodies, with orwithout an adjuvant, followed by additional injections of the protein atbiweekly or longer intervals and monitoring the level of antisera. Formonoclonal antibodies, splenocytes may be isolated, immortalized andscreened. Those hybridomas which produce antisera which can distinguishbetween the conformers are expanded. A library is produced of antibodiesthat distinguish between the two conformers. The antibodies may then beused to isolate each of the conformers and assay for the differentconformers in hosts, using physiological samples appropriate to thenature of the protein.

[0040] Signal sequences may also be rated by analysis of crosslinkingpatterns generated when truncated transcripts encoding those signalsequences at the 5′ end of the authentic coding region of interest areexpressed by cell-free translation and subject to chemical crosslinkingincluding but not limited to lysine and cystiene specific cleavable anduncleavable crosslinkers, with analysis of the crosslink patterns byimmunoprecipitation and polyacrylamide gel electrophoresis in sodiumdodecyl sulfate and subsequent autoradiography.

[0041] Different conformers may be screened by employing matrices ofdifferent oligopeptides and/or oligonucleotides. See, for example, U.S.Pat. Nos. 5,631,734; 5,856,102 and 5,919,523. These matrices areavailable commercially and can be prepared in relation to a particularbinding pattern. In this way, one can add a physiological sample and seewhich of the conformers are present and estimate the amount of each. Onemay also use the matrix to isolate particular conformers by theirbinding affinity to the matrix. The matrix and antibodies may be used inconjunction to confirm the other assay, isolate the conformer, usedtogether in the same assay. In assays by themselves or in conjunctionwith other affinity binding assays, the antibodies may be labeled with adetectable label, e.g. fluorescer, luminescer, phosphorescer, enzyme,radioisotope, and the like. Numerous protocols are available fordefining specific epitopes, by which the conformers may bedistinguished. In some instances, it may be desirable to use two or moreantibodies, where the conformer may be defined by steric inhibition ofbinding, different affinity constants, or the like.

[0042] By virtue of the fact that the conformers can be distinguished byoligomers, particularly of oligopeptides, these oligopeptides may servefor identifying the different conformers. The oligopeptides can be usedin competitive assays for identifying other oligopeptides which competefor the site or other compounds, particularly small organic compounds,natural or synthetic, of less than 5 kDal, usually less than about 2.5kDal, which bind to the conformer. These compounds may then be used inturn to identify other compounds having greater affinity for the site.In this way drugs may be identified that are specific for one conformer,as compared to other conformers. The various binding entities may beused in assays, where the entity may be labeled to identify binding tothe conformer. The assays may be homogeneous or heterogeneous.

[0043] If desired, one may use random mutations of the wild-type and/orchimeric gene to express the resulting gene. The protein may be analyzedfor similarities and differences with the parent gene. Where the effectof the mutation is to change the conformer from one conformation toanother or to change an epitope, as determined by antibody binding. Orto generate differences in crosslinking pattern or conformation asscored by altered reactivity to chemical modifying agents undernon-denaturing condtions, including but not limited to N-sulfobiotin,trinitrobenzenesulfonic acid (TNBS), N-ethyl maleimide (NEM). Genes andthe proteins, which they express from human patients or animals, where amutation is naturally occurring or a result of mutating a gene, may becompared with the randomly mutated gene and its expression product in amammalian host. In this way, disease states resulting from mutationsthat lead to different conformers may be diagnosed and treatmentsdeveloped.

[0044] The conformers can be used for biomedical purposes in aiding inthe diagnosis and treatment of patients. The conformers serve todifferentiate between individual hosts, who produce the conformers indifferent ratios, normally or under different environments. One of theenvironments is the presence of a compound, particularly a drug, and thebinding affinity and/or modulation of activity of a targetphysiologically active compound, usually a protein, such as a membranereceptor, channel, enzyme, transcription factor, housekeeping protein,cytoskeletal protein, membrane protein, and the like. The conformers maybe mobile or immobile proteins, being bound to membranes or free insolution. A compound would be mixed with the different conformers, inthe same or different vessel, and the binding of the compound to theconformer determined. The determination may be made in a variety ofways, either competitive or non-competitive. The amount of compoundremaining in the solution may be determined, where a reduction in amountwould be indicative of the binding of the compound to the conformer.Alternatively, a labeled compound which binds to the conformer may beused, where interest in binding is as to a particular region of theprotein, such as in the case of enzymes and receptors, as well as otherproteins where particular sites are associated with biological activity,e.g. G-proteins, transcription factors, DNA binding proteins, etc.

[0045] The conformers are also useful in determining differences inactivity in relation to their physiological activity. The bindingaffinity for their binding partners can be determined in assays, eitherhomogeneous or non-homogeneous, which allows for evaluation of the levelof activity of a mammalian host in relation to the proportion of the twoconformers. Again, numerous protocols are available for detectingbinding between a ligand and a receptor or two or more proteins involvedin complex formation. The two proteins may be labeled with a fluorescerand an energy receptor, so that binding of the two proteins togetherwould reduce the level of emission at the wavelength of the fluorescerand increase the emission at the level of the energy acceptor.Alternatively, one may bind one of the proteins to a surface and thelevel of binding of the other protein to the bound protein determined byhaving the second protein labeled. Alternatively, one may bind each ofthe proteins to different particles, where one particle produces acompound, which activates the other particle, such as LOCI. Where thelabeled entity is small, such as an oligomer or small organic molecule,a fluorescer may be used as the label and fluorescence polarizationemployed for detection. Illustrative of assays are the assays describedin U.S. Pat. Nos. 5,989,921; 4,806,488; 4,318,707; 4,255,329; 4,233,402;and 4,199,559.

[0046] In determining the presence of conformers when screeningphysiological samples, the samples may be blood, cells, csf, saliva,urine, hair, etc. The sample may be subject to pretreatment, such asadding citrate to blood, coagulating and separating erythrocytes,dilution, extraction, etc. Thus, the conformers may be identified asbeing associated with particular indications, which may relate todiseases, response to drugs, physical performance, cellulardegeneration, apoptosis, etc.

[0047] Illustrative of the power of the subject invention is theinvestigation of prions. It is found that by varying the signal sequenceone can obtain the natural conformation ^(sec)PrP, and two otherconformations, ^(Ntm)Prp and the neurodegenerative ^(Ctm)Prp, in varyingamounts. This aids in the determination of the mechanism of the changein the proportion of formation of the neurodegenerative form as comparedto the wild-type form. In an analogous manner, one may vary the signalsequences for other proteins having signal sequences, where the proteinis suspected of being associated with a diseased state or loweredperformance. One could then determine whether the protein can beproduced in varying conformations. If different conformers are found, asdescribed above the presence of the different conformers is establishedin healthy normal patients as compared to patients who have the diseaseor reduced performance. Drugs would then be screened as described aboveagainst the undesired conformer.

[0048] In addition, by using different signal sequences one caninvestigate the basis for the change in proportion between the desiredconformer and the undesired conformer. In conjunction with, in additionto or in place of, one can use in vitro lysates for investigating therole of ER associated proteins with the formation of the conformers.Microsomes are prepared lacking all but the essential proteins fortranslocation. The individual proteins may then be replaced individuallyor in combination to determine the effect of the presence of theprotein(s) on the formation of the conformers. Once the protein(s)involved in the formation of the conformers is determined, one canscreen healthy and abnormal patients for the presence of the protein(s)involved in the formation of conformers and determine the presence ofmutations or different conformers. In this manner, one not onlyelucidates the mechanism by which proteins fold in the translocon, butalso establish new targets for therapies.

[0049] For the cell-free protein translation mixture, various systemsmay be employed (Erickson and Blobel, Methods Enzymol. 96:38-50 (1983);Merrick, Methods Enzymol 101:606-615 (1983)). Illustrative are wheatgerm extract and rabbit reticulocyte extract, available from commercialsuppliers, e.g. Promega (Madison, Wis.), as well as high-speedsupernatants thereof. Other references include U.S. Pat. Nos. 5,998,163;5,998,136 and 5,989,833. Such systems include, in addition to thecell-free extract containing translation machinery (tRNA, ribosomes,etc.), an energy source (ATP, GTP), and a full complement of aminoacids. Methods known in the art are used to maintain energy levelssufficient to maintain protein synthesis, for example, by addingadditional nucleotide energy sources during the reaction or by adding analternative energy source, e.g., creatine phosphate/creatinephosphokinase. The ATP and GTP present in the standard translationmixture will generally be at a concentration in the range of about 0.1to 10 mM, more usually 0.5 to 2 mM. Generally, the amount of thenucleotides will be sufficient to provide at least about 5 picomolar ofproduct, preferably at least about 10 picomolar of product.

[0050] Lymphocytes also form PrP and may be used as a screen for theeffect of agents, e.g. compounds as candidate drugs or as a screen forresponse of an individual to changes in the environment or physicalinsults. The lymphocytes would be subjected to a change in theenvironment, which could be a chemical change, e.g. addition of acompound, a physical change, e.g. change in pH, etc., where thesensitivity of the particular cells to the change would be determined asa measure of the propensity of the person to respond to theenvironmental change by changing the nature of the PrP. By determiningthe response to changes in the environment, which could includepollutants, pesticides, etc., one can determine whether the compoundsare a general threat or only affect idiosyncratic people. The cells mayalso be used to determine whether a patient who is symptomatic for aneurodegenerative disease has a Ctm-PrP related disease by determiningthe presence and/or level of Ctm-PrP in the lymphocytes as compared tonormal individuals. Numerous assays may be employed as described hereinto determine the presence and level of Ctm-PrP. By detecting apropensity for neurodegenerative disease, the patient could be directedaway from activities or exposure to compounds that might increase theprobability of the neurodegenerative disease. In addition, the effect ofcompounds on Ctm-PrP may be used as a substitute in relating theactivity to other diseases that respond in an analogous way. The cellsmay also be used for individual patients to evaluate the response of theindividual to various drugs, determining the effect of the drug on theproduction of Ctm-PrP.

[0051] As described above, many proteins have different translocationaloutcomes. Proteins known to have alternatives in the translocationalpathway, such as existing in two different conformers, the presence ofdifferent methionines that may be selected as the f-Met, resulting in adifferent signal sequence, having hydrophobic regions of from about 15to 20 amino acids in the chain, which can be putative transmembranesequences, but are available as secreted proteins or are transmembranethat are secreted can be used as markers for the effect of compounds ontranslocational outcome. Illustrative proteins include ductin,calreticulin, MDR-1, and PrP.

[0052] Numerous proteins are associated with the ER in the process oftranslocation, frequently being part of the ER and associated with thechannel through which the nascent protein is transported to the ERlumen. These proteins include the ribosome, proteins of the signalrecognition particle (SRP), the heterotrimeric Sec61 complex with α, β,and γ-subunits, translocating-chain-associated membrane protein (TRAM),signal peptidase (a complex of five proteins), oligosaccharyltransferase (a 3-protein complex), ER lumenal proteins, including BiP,GRP94, calnexin (CNX), ERGIC-53, protein disulfide isomerase (PDI),Erp57. Erp72, chaperones, such as Hsp 90, hsp 47, Hsp 60 or GroELfamily, Hsp 70 or DnaK family, Hsp100 or Clp family, and heat shockcognate 70, tapasin, microsomal triglyceride transfer protein,protective protein/cathespin A, β-catenin, egasyn, co-chaperones, suchas proteins from the DnaJ family, enzymes, such as uridine5′-diphosphate (UDP) - glucose:glycoprotein glucosyltransferase (GT),glucosidase II, prolyl-hydroxylase and carboxylesterase.

[0053] The ancillary proteins associated with the ER and translocationof the translocation product may be removed from the lysate by methodsdescribed in the literature. (Gorlich, et al., (1992) Nature 357, 47-52;Gorlich and Rapoport, (1993) Cell 75, 615-630; and Hanein, et al.,(1996) Cell 87, 721-732.

[0054] By varying the signal sequence in proteins of interest, one canprepare genes for expression in mammalian hosts. Methods of replacing aDNA sequence to provide a chimeric gene are legion today. See, forexample, Sambrook, et al. (1989), A Laboratory Manual, Second edition,Cold Spring Harbor Press. Briefly, the DNA for the gene is isolatedknowing the amino acid sequence and using degenerate probes. The DNAsequences, which bind to the probes, are isolated and sequenced to seethe presence of a sequence coding for the protein of interest. If onewishes to avoid the presence of introns, the mRNA may be isolated,reverse transcribed and amplified using PCR. The signal sequence ineither situation may be replaced with a different signal sequence byamplification using one pair of primers, with one primer having a signalsequence at its 5′-terminus joined to a sequence complementary to thesequence of the gene contiguous with the native signal sequence theother primer complementary to the first primer. A second set of primerswill provide the other terminus of the DNA sequence. Depending on thesize of the gene, one may select a convenient restriction site forlinking the modified 5′-terminus DNA with the remainder of the codingsequence. Various techniques are available and each gene will have anobvious selection of protocols to enhance the convenience of theparticular synthesis.

[0055] Once the gene is produced it may be introduced into one ofnumerous commercially available vectors and cloned and/or an expressionvector may be employed having a transcriptional regulatory region 5′ ofthe sense strand to provide for expression. By using mammalian cells,the conformers from the different constructs can be produced andisolated and assayed as described above. In this way, significantamounts of the different conformers may be isolated.

[0056] In an initial stage, one may wish to concentrate the conformersusing different separation techniques, such as HPLC, capillaryelectrophoresis, affinity chromatography, etc. The initial separationmay solely serve to enhance the concentration of the conformers in aparticular fraction or may provide for separation of the conformers. Inthe former case, one would need further separation of the conformers,using techniques, which have been described above. Various separationmedia may be employed, involving ion exchange, sieving media, affinitymedia, etc., using different eluents to establish procedures forseparation and isolation of the different conformers. Various procedureshave been developed and each set of conformers will use protocols thatoptimize the separation with minimum denaturation. These protocols maybe readily identified by those of skill in the art of proteinpurification. The purified conformers may then be used for assays, forx-ray crystallography, to identify differences in structure, the aminoacids associated with the different epitopes, the interactions withproteins with which the wild-type protein is associated, as well asother proteins to which the conformer may bind, and the like.

[0057] Once having the gene for the chimeric protein, the gene can beused in gene therapy, mutating laboratory animals, random mutation todetermine the effect of mutations on folding, in expression constructsand single cell or organism host for large scale production of theprotein, and the like. Laboratory animals include rodents, lagomorpha,birds, canine, feline, porcine, etc.

[0058] In some instances, one may create viral constructs, where thechimeric gene is introduced into a viral carrier for introduction intocells, either randomly or where the virus has a tropism, into cells forwhich the virus is tropic. Where the gene has a beneficial effect, evenin the presence of the other conformer, the presence of the constructmay serve to alleviate the symptom resulting from the native conformer.Alternatively, the introduction of the non-native conformer will allowan evaluation of the effect of the non-native conformer on thephysiology of the cell, tissue, organ and host.

[0059] Of particular interest is the introduction, preferablyreplacement of the wild-type gene with the chimeric construct comprisingthe wild-type structure and a different signal sequence that is known toresult in a different conformer. By using homologous recombination withgerm cells or a nucleus that is subsequently transferred to a germ cell,one can provide for the expression of the desired conformer(s). Whereheterozygosity is involved, the progeny having the chimeric gene may bemated to obtain homozygous progeny. In this way, one can investigate therole of the conformer(s), whether the location(s) to which the proteinis transported changes, the effect of such variation in location, theeffect of the different epitopes or topography on the function of theprotein, and the like. In addition, the chimeric mammal may be used toscreen compounds for their biological effect on the conformer, so thatdrugs which are directed to a particular indication, which involves theconformer, either directly or as a result of the conformer being in thepathway, can be evaluated. The animal models may be used todifferentiate the effects on the wild-type conformer and otherconformers that can be formed.

[0060] In situations where the non-native conformer is a dominantnegative allele, the loss of activity can be monitored as to the effecton the physiology of the host. The effect on the host can be compared todisease states, where similar indications are observed. Treatments canthen be evaluated by employing drugs having known function associatedwith the indication, compounds known to bind to the wild-type allele orother treatment to evaluate the effectiveness of the treatment. Cellshaving the unnatural conformer may be screened for the effect of theunnatural conformer on the expression of other proteins. By convertingthe MRNA to cDNA of such cells, one can by using various subtractiontechniques known in the literature, between the cells without theunnatural gene and the cells with the unnatural gene, determine theeffect of the unnatural gene on the expression of other proteins.Alternatively, one may express the various proteins and compareelectrophoretic, mass spectrophotometric or HPLC patterns to determineif the different conformers affect the expression pattern of the host.

[0061] Following the procedures described in the above description andin the experimental section, or other functionally equivalentprocedures, the subject invention allows for investigation of diseasesassociated with the prion protein and analogous diseases, particularlydiseases associated with topological modifications as in Alzheimer'sdisease. As shown, by preparing expression constructs for expression ofthe native PrP, the Ntm-PrP and the Ctm-Prp, one can modify cells andanimals to produce the different forms of the protein. In this way, onecan study in culture and in vivo the effect of the addition of variouscandidate agents and determine the change in the formation of thedifferent conformers in the presence of the agents in culture and invivo. The subject proteins allow for the controlled presence of thedifferent conformers and a study of the etiology of the disease, theother entities involved in the etiology of the disease, by doingexpression analysis of the effect of the different conformers onexpression in the same and different cells and on the physiology of thehost. Mouse models may be developed with the construct in the presenceor absence of the native PrP gene, since the host gene may beknocked-out and replaced with the chimeric gene. Alternatively,mutations may be introduced into the PrP gene that result in amodification of folding and a modification in the dependence on orinteraction with proteins associated with translocation. Thus, thesignal sequences, either native or chimeric to the gene, may be mutatedand the effect on translocation-related proteins determined.

[0062] Uses of the Invention

[0063] The invention finds use at the molecular, cellular and organismallevels (i.e. in elucidating, diagnosing or treating a disease).Following are provided specific applications.

[0064] 1. Using the described invention, it would be possible tomanipulate antibodies to increase their functions and uses. By signalsequence replacements, alone and in conjunction with fractionated andreconstituted or variant membrane and cytosols in cell-free translationsystems, the pathway of folding of an immunoglobulin could be altered ineither subtle or profound ways, without actually changing the amino acidsequence of the final product itself. Thus, it would be possible toachieve variant immunoglobulins in which the binding constant wereincreased or decreased incrementally, and immunoglobulins with changesin their effector functions (e.g. the binding specificity of their Fcregion). Current approaches to achieve these ends (e.g. by site-directedor random mutagenesis of the authentic immunoglobulin chains) have thedisadvantage of changing not only the folding pathway but also theprimary structure of the molecule itself. Our approach would, in effect,dissociate this two types of changes, allowing one without the other,with diagnostic and/or therapeutic utility. The signal sequencevariations could also be applied to transfected mammalian cells,allowing medium to be harvested and screened for conformational andfunctional differences.

[0065] 2. Most hormones, cytokines, and growth factors have beenimplicated in a wide range of functional behaviors, and in some cases,activation of multiple signaling pathways. For example, leptin, firstdiscovered as a hormone that signals satiety, has been found to serve asan independent central regulator of bone density, and to be implicatedin wound healing and control of blood pressure. Some of these functionsclearly occur in the absence of the others, and most obese individualsare leptin resistant, without having disorders of bone density,suggesting that the leptin they make is not able to serve the satietypromoting function, but is able to function in maintenance of bonedensity. The conventional assumption has been that this sort ofheterogeneous action is achieved through diversity in receptors orreceptor function. However, diversity of ligand conformation, asdemonstrated in the examples from PrP and prolactin shown here, couldaccount for some or even most of the heterogeneity of outcomes, in thecase of leptin.

[0066] By allowing individual conformers to be identified, andpharmaceuticals to be developed to increase or decrease the expressionof individual conformers, or to block action of one conformer oranother, it should be possible to affect gene expression in ways thatpromote some functions of hormones over others. It should also bepossible to stratify patients into subsets based on the mix ofconformers that is manifest with their particular genotype andphenotype. This would in turn, allow different treatments to beadministered to different individuals taking into account the mix ofconformers they are expressing at that particular point in time, usingdifferent pharmaceutical agents designed or shown to be most efficaciousfor a particular conformer mix or other conformer-related subset.

[0067] Thus, in the case of leptin, swapping of signal sequence, andsynthesis in various combinations of translation systems containingfractionated cytosol and fractionated and reconstituted or variantmembranes, and subsequent screening programs should make it possible togenerate forms of leptin that would promote bone density without affectson satiety or wound healing, or vice versa. Likewise, forms of leptinthat would be more active at appetite suppression and thereby overcomeleptin resistance, the most commonly observed phenotype in obese humans,could be generated. Conversely, small molecule pharmaceuticals could bedeveloped, by screening for their effect on individual subsets of leptinconformers, or for their affect in altering the mix of leptin conformerssecreted. Thus it would be possible to develop drugs that haveconformer-specific or selective effects, thereby blocking or enhancingthe effects of conformers, or blocking or enhancing the body's abilityto make a particular mix of conformers. Half-life, association withother molecules, secretion kinetics are parameters besides affinity forparticular receptors that could be altered by signal sequencemanipulations without altering in any way the sequence of the mature,authentic protein. This approach would apply also to secretory andintegral membrane enzymes, where substrate turnover, rather thanreceptor affinity is the relevant functional parameter.

[0068] 3. This approach can be combined with transgenic technology tointroduce variant genes differing in their signal sequences, intowild-type and knock-out mice (lacking an endogenous gene). In thismanner it will be possible to screen for functions of complex secretoryand integral membrane proteins that currently are not known. Thus it isknown that 95% of the wild-type Cystic Fibrosis Transmembrane Regulator(CFTR) is degraded immediately upon synthesis. The conventionalassumption is that this represents “errors” in biogenesis. Analternative interpretation, for which the present invention has utility,is that these 95% represent the sum total of many alternativeconformations that are not needed by the cell at one particular time,but may be rescued from degradation at particular times in development.Since the signal sequences of CFTR are not cleaved, the goal of alteringfolding without introducing mutations must proceed exclusively byregulation in trans rathe than in cis. Thus, expression of CFTR intranslation systems complemented with fractionated and reconstituted orvariant membranes and fractionated and reconstituted cytosol, asclaimed, would enhance one versus another conformation, which would inturn be scored and catalogued by monoclonal antibody reactivity,chemical modifications, crosslinking and other properties, and thenintroduced into transgenic animals and screened for novel phenotypes anddisorders. Similar approaches can be taken for each of the channelforming and receptor forming membrane proteins, including both singleand multispanning with respect to the ER and/or other internal and/orplasma membranes.

[0069] 4. The most difficult to currently treat diseases in humans aredisorders of signaling as typically manifest by hormone, growth factor,cytokine and receptor resistance (also known as desensitization or downregulation). Many different mechanism of resistance have beenidentified, whether most cases of most diseases are due to themechanisms identified or due to other as yet unknown mechanisms remainsto be determined. Based on our studies with PrP as summarized in ourpublished and unpublished work, we believe that conformationaldysregulation (the wrong mix of conformers being made or being allowedto exit from the ER) is a central mechanism of disease pathogenesis.This can be demonstrated by raising monoclonal antibodies to secretoryproteins and demonstrating that individual subsets of the secretoryproteins are present (or absent) in a disproportionate manner in adisease state or in subsets of patients with a disease state. The majorgenes for the major diseases afflicting humans include a large number ofsecretory and membrane proteins, which could be screened for theinvolvement of conformational dysregulation in their pathophysiology.Diabetes, hypertension, hyperlipidemia, obesity, osteoporosis,degenerative joint disease, cancer, Alzheimer's disease, and psychiatricdisorders are just a few examples already implicated in this fashion.

[0070] Application of the Invention to the Research and Treatment ofDisease

[0071] As listed above, a number of important medical conditions involvegenes for which conformational regulation is likely to be an importantdimension of physiological and pathophysiological function. Here it isdescribed how the invention is applied to better understanding specificdiseases.

[0072] First, key disease-related genes that are secretory and integralmembrane proteins would be analysed in cell-free systems and intransfected mammalian cells, to characterize the class in which theirsignal sequences fall. The conformational heterogeneity of the nativeproteins will be catalogued both in vitro and in vivo, including insamples from mice and humans (e.g. from blood), if available, usingmonoclonal antibodies, chemical modifications and co-association withother proteins inside the cell and in the medium, as the means ofscoring conformation.

[0073] Second, attempts will be made to skew the conformational mixsynthesized, allowing minor and transient conformers to be magnified andstabilized and therefore more readily detected and characterized, sothat they can be distinguished from the normally dominant conformers.This can be done in two general ways, namely by swapping signalsequences that are cleaved or by expressing the proteins in fractionatedand reconstituted systems that modify the native conformer mix throughactions (or their absence) in trans.

[0074] Third, once the mix of conformers that define the keydisease-related proteins has been characterized, samples from patientsrepresenting the diversity of the natural history and phenotypic classesof the disease in question, will be screened and categorized withrespect to the heterogeneity of conformers of these proteins observed.From this analysis it will be possible to identify i) the conformersimplicated in disease; ii) changes in conformer mix that precede actualdevelopment of disease, iii) changes in conformer mix that stratifyindividuals with respect to disease progression, complications and otheraspects of natural history, including increased or decreased risk ofdrug efficacy, side effects and other reactions. Note that these are allgoals of conventional proteomics programs which will be missed by thoseprograms because they are not aware of the evidence, submitted insupport of the subject invention, for conformational heterogeneity ofproteins. Hence they are not looking for alternate conformers of theprotein in question. Furthermore, without the subject invention, thereare currently no means of systematically identifying, magnifying, andcharacterizing these changes in conformers, without which it isimpossible to determine their distribution or significance inpopulations at risk of, or afflicted with, particular diseases.

[0075] Fourth, with a variety of valuable information on diseaseassociation of conformers in hand from 1-3 above, it is possible todevelop assays as outlined previously to screen for agents that modifythe conformer mix in a way that minimizes undesired conformers andmaximizes those that are protective or not associated with diseaseprogression, drug toxicity, etc. Likewise, agents that block undesiredconformers selectively, or that enhance the action of desired conformerscan be sought through high throughput screens of large compoundlibraries, as well as through conformer-specific rational drug design.

[0076] Specific Applications to Some Major Diseases are Outlined Below.

[0077] Diabetes mellitus: The key genes for insulin, the insulinreceptor and the many members of the family of glucose transporters allhave cleaved or uncleaved signal sequences that make them amenable tothis analysis. Furthermore, a key pathophysiological process impactingon patient care is the syndrome termed insulin resistance. Usually thisis attributed to disorders in receptor function, but in most cases, thebasis for the disorder is unknown, and the possibility that the disordercould derive from the ligand rather than the receptor, cannot be ruledout. In this case, the ligand is insulin, and the aberrant conformationthat impairs insulin-mediated signaling could be a property of eitherinsulin or the insulin receptor or individual members of the family ofglucose transporters, which could be identified in the manner described.Finally, the huge heterogeneity of individual phenotype at any giventime, and in natural history over time, which has remained elusivedespite massive research efforts directed at conventional genomic andproteomic analysis, is strongly suggestive of disordered conformatics,amenable to the subject invention.

[0078] Hypertension: A number of key genes in control of blood pressureencode secretory and integral membrane proteins including the epithelialsodium channel (EnaC), angiotensinogen, the angiotensin receptors,renin, the enzymes of steroid biosynthesis involved in synthesis ofaldosterone and other steriods, and the alpha and beta adrenergicreceptors, to name a few. The pathophysiology of hypertension remainslargely mysterious, hence the possibility that conformer dysregulationplays a role in its etiology and pathogenesis cannot be excluded in anyway. Clinical epidemiological and observational studies clearly indicatethat patients are heterogeneous not only with respect to the nature oftheir hypertension-inducing state (e.g. salt sensitive vs saltinsensitive; increased sympathetic activity, etc.) but also with respectto their sensitivity to individual classes of drugs, side effects ofthose drugs, and ability to tolerate the drugs.

[0079] Obesity: The key genes involved in the control of obesity,including leptin and its receptors, and the various obesity relatedgenes identified to date including the melanocortin and mahoganyreceptors, the agouti protein, etc. all contain cleaved or uncleavedsignal sequences. Furthermore, most obese individuals are leptinresistant, consistent with the hypothesis that conformer differences ofleptin mediate its diverse physiological functions (e.g. in satiety,maintenance of bone density, promotion of wound healing, and regulationnof blood pressure).

[0080] Cancer: Conceptually, cancer is a disorder of signaling pathwaysinvolved in cell growth and proliferation and the physiological controlsover these processes, including a host of diverse apoptotic triggers andantiapoptotic factors. Many of these are secretory or membrane proteinswith cleaved or uncleaved signal sequences. In many cases, cancer can beaffected, both positively and negatively, by a host of secreted growthfactors and cytokines, which also generally have signal sequences. Ahost of data supports the notion that many of these factors aremultifunctional, or more precisely, are associated with both promotingand inhibiting particular signalling pathways. A present conundrum inthe field is understanding how one factor can bring about one action atone point in time, and yet bring about a very different action atanother time. A switch from one to another conformer resulting inaltered receptor interaction, signal transduction, half life,associations etc., could be central to one or more pathways ofcarcinogenesis and phenotypic variation in the development, immunesurveillance, presentation and progression of cancer, or in response toits treatment.

[0081] Osteoporosis: The hormones involved in the regulation of bonedensity, including leptin discussed earlier, and others such asparathyroid hormone, osteoprotegenin, and their receptors, all havesignal sequences, some cleaved, some uncleaved, rendering them amenableto analysis by the subject invention. The physiology of bone density iscomplex, poorly understood and subject to a variety of confoundingparadoxes - the same agent that promotes bone density in somecircumstances can result in bone loss under others. Conformationalheterogeneity in response to signaling changes is a potential basis forthese observations, with broad potential for diagnostics andtherapeutics.

[0082] Neurodegeneration: Including prion and alzheimer's diseases,spinal cord injury, and stroke, are areas of perhaps the greatestpotential for diagnosis and therapy involving conformatics. Prionprotein, the gene product responsible for prion diseases was the firstprotein for which conformational regulation was demonstrated. In thiscase, conformational regulation is manifest as topologicalregulation—the three detectable conformers differ in transmembranetopology, making them relatively easy to identify and distinguish.Amyloid Precursor Protein, the gene product whose aberrant metabolitehas been implicated in Alzheimer's disease, is an integral membraneprotein which has features in common with the prion protein, including acleavable signal sequence. Netrins, semiphorins and other genesimplicated in axonal guidance (and therefore central to recovery fromspinal cord injury), have paradoxical activities consistent with thehypothesis of multiple conformers.

[0083] Coronary artery disease: Apolipoprotein B is a complex,multifunctional secretory protein involved in low density lipoproteinmetabolism and a prime candidate for conformational heterogeneity.Essentially every integral membrane channel and receptor protein in thebody have uncleaved signal sequences amenable to fractionation asdescribed elsewhere.

[0084] Chronic obstructive pulmonary disease: The Cystic FibrosisTransmembrane Regulator (CFTR) is a multispanning integral membraneprotein discussed previously, whose biogenesis suggests a more complexfate that is generally accepted. One reason for the difficulty inacceptance of the hypothesis that degraded chains of CFTR representalternate conformations not needed at that point by the cell, ratherthan true misfolded chains, is that our evidence in favor ofconformatics remains largely unpublished. While those alternativefunctions remain unknown at the present time, it has been speculatedthat, the known function of CFTR as a chloride channel affects the saltenvironment needed for anti-microbial peptides such as the defensins,needed for innate immunity against pathogenic microbes. Thus,conformational dysregulation in which chloride channel function is lostmay confer increased susceptibility to infection in the lung and othertissues, as is observed in a wide range of pulmonary and otherdisorders. By using fractionated cell-free translation and translocationsystems, as described in this invention, in combination with availablescoring systems including monoclonal antibodies, chemical modificationfingerprints, crosslinking and association analysis including sucrosegradient studies, we can catalogue the alternative conformations ofCFTR, MDR genes, and other relevant transporters and determine whetherthese alternatives are utilized to a greater or lesser extent inparticular subsets of patients with infectious and other disorders.

[0085] Psychiatric disorders: As with neurodegeneration, functionaldisorders of the brain are likely to involve disturbances in signalingand/or signal transduction. Since receptors on the cell surface,including the family of G protein-coupled receptors, are generallyresponsible for cell to cell signaling and generally have signalsequences, cleaved or otherwise, these classes of proteins are highlylikely to be candidates for conformational regulation.

[0086] Taken together, the examples cited above demonstrate the broadnature of this invention and its applicability to a wide range ofdisorders in which a signal sequence containing protein or receptor isinvolved.

[0087] Relevant manuscripts accompany this application, were submittedas part of priority application Ser. Nos. 60/171,012 and 60/172,350, andare included by incorporation by reference, as if they were specificallyset forth herein. These manuscripts are identified by their title andfirst author: Rutkowski, et al., “A New Role for the Signal Sequence inTranslocational Regulation”; Hegde, et al., “Transmissible and GeneticPrion Diseases Share a Common Pathway of Neurodegeneration” (publishedin Nature (1999) 402:822-826); and Lingappa, et al., “ConformationalControl Through Translocational Regulation: A New View of Secretory andMembrane Protein Folding.”

[0088] The following examples are offered by way of illustration and notby way of limitation.

EXAMPLES

[0089] Materials

[0090] Rabbit reticulocyte lysate (RRL) and dog pancreatic roughmicrosomes were prepared and used as described (Hegde and Lingappa, Cell(1996) 85:217-228, and references therein). Mouse brain microsomes wereprepared in the same manner as canine microsomes. PK was also preparedas described (Hegde et al, Cell (1998) 92:621-631. Anti-prolactinantibody was purchased from USB (Cleveland, Ohio) and 3F4 monoclonalanti-PrP antibody was a gift from the Prusiner laboratory. Anti-PDI waspurchased from StressGen (Victoria, BC). Disuccinimidyl suberate (DSS)was from Pierce (Rockford, Ill.). Saponin was from Calbiochem (La Jolla,Calif.) and was dissolved as a 20% w/v stock in water, adjusted to 10 mMHepes, pH 7.2, and the contaminants removed by passage over a 1.5 mlcolumn (per 10 mls saponin) of SP-sepharose fast flow (AmershamPharmacia; Piscataway, N.J.) and a 2 ml column of Q-sepharose fast flow.A clone encoding yeast prepro-alpha factor was provided by Tom Rapoport.

[0091] Fractionation of Reticulocyte Lysate.

[0092] The process described herein is for the preparation of a modifiedin vitro translation system derived from the presently available rabbitreticulocyte lysate (RRL) translation system. The RRL translationsystem, originally developed by Jackson and Hunt (Methods Enzymol (1983)96:50-74), offers a mammalian based extract competent for translation ofmessanger RNA (either synthetic or native). Furthermore, whensupplemented with microsomal membranes, the biogenesis of secretory andmembrane proteins can be reconstituted. By fractionating the RRL intodefined components, we have extended the flexibility and potential usesof the in vitro translation system. These advances have severaladvantages for addressing a variety of biological phenomena (see below).Briefly, RRL is separated into native ribosomes, and a soluble proteinfraction (S-100). The S-100 is further fractionated by anion exchangechromatography on DEAE sepharose. The flow-thru (containing all of theglobin) is discarded, and the column step-eluted with 300 mM KCl. Theeluate (which contains the relevant translation factors) is concentratedby ammonium sulfate precipitation followed by dialysis. This DEAEfraction, along with the ribosomes can reconstitute translation withessentially equal efficiency as the starting RRL. The following is thedetailed protocol for preparation of the fractions from 1 ml of RRL. Itcan easily be scaled up as necessary. Column buffer is 20 mMTris-Acetate, pH 7.5, 20 mM KCl, 0.1 mM EDTA, 1 mM DTT (added fresh),10% v/v Glycerol. Elution Buffer is same as Column Buffer, but with 300mM KCl. Dialysis Buffer is 20 mM Hepes-KOH, pH 7.5, 100 mM KOAc, 0.5 mMMgOAc, 0.1 mM EDTA, 1 mM DTT (added fresh), 10% v/v Glycerol.Fractionation protocol: i) RRL is prepared according to previouslypublished protocols (Jackson and Hunt, supra), except that it is notdesalted. Briefly, blood cells from an anaemic rabbit are washed severaltimes, and the cytosol released by hypotonic lysis. The unlysed cellsand ghosts (cells which have lysed and released their cytosol) areremoved by centrifugation. The supernatant is the RRL used in thisfractionation. ii) RRL is adjusted to 1 mM CaCl₂, digested with 150 U/mlmicrococcol nuclease for 10 minutes at 25° C., and the reactionterminated by the addition of EGTA to 2 mM. The RRL is chilled to 0° C.on ice, and all subsequent procedures done at 4° (in the cold room) oron ice. iii) The nucleased RRL is centrifuged for 20 minutes at 100,000RPM in thick-walled polycarbonate tubes (1 ml capacity) in a TL-100.2rotor at 4° C. The supernatant (S-100) is removed to a pre-chilled 2 mleppendorf tube on ice. The pellet is rinsed once in Dialysis Buffer, andresuspended in 100 μl of dialysis buffer. The ribosomes are then frozenin aliquots and stored at −80° C. They are stable for at least one yearwithout any loss of activity. Avoid excessive freeze-thaws, although itappears stable to at least 2-3 uses without any problems. iv) The S-100is diluted with an equal volume (1 ml) of column buffer and applied to a3 ml DEAE sepharose column. The column is washed with additional columnbuffer until the last traces of the bright red globin have flowedthrough. The column is then step eluted with 10 mls of Elution Buffer,and eluate collected. To the 10 mils of eluate, 5.6 grams of solidammonium sulfate is added slowly with constant stirring (bringing it to˜80% saturation). Stir at 4° C. (or on ice) for an additional 20 30minutes after the last of the ammonium sulfate is added. Sediment theprecipitate by centrifugation (10,000×g for 15 minutes), remove anddiscard the supernatant, and dissolve the pellet in at most 1 ml ofdialysis buffer, preferably 0.5 ml (it goes into solution readily withonly gentle mixing). Dialyze against 500 ml dialysis buffer to removethe residual anunonium sulfate (with one change of buffer if desired) at4° C. for 8-12 hrs. The sample is recovered from the dialysis tubing,frozen in aliquots, and stored at −80° C. It is stable for over 1 yearwith no noticeable loss of activity. Avoid excessive freeze-thaws,although it appears stable to at least 2-3 uses without any problems.

Example 1 Prion Protein

[0093] A dramatic example of a substrate with complex and highlyregulated translocation is the prion protein (PrP), a 35 kD brainglycoprotein involved in the pathogenesis of several neurodegenerativedisorders (Prusiner (1997) Science 278, 241-251). PrP is simultaneouslysynthesized in three alternate topological forms at the ER (Hegde etal., (1998) Science 279, 827-834). One of these forms, termed ^(sec)PrP,is fully translocated across the ER membrane, and is the predominantform observed in vivo. By contrast, the other two forms of PrP are madeas singly-spanning membrane proteins in opposite orientations witheither the N- or C-terminus in the ER lumen (termed ^(Ntm)PrP and^(Ctm)PrP, respectively). PrP biogenesis appears to involve multiplesteps leading to at least three distinct and assayable endpoints, ittherefore was used as a sensitive probe for any potentialsubstrate-specific effects of N-terminal signal sequences. In thefollowing experiments, the native signal sequence of PrP was replacedwith the well-studied signal sequences of the secretory proteinspreprolactin and pre-beta-lactamase (FIG. 1A). The chimeric proteins(Prl-PrP and βL-PrP, respectively) were compared to native PrP for theirability to be targeted, translocated, and synthesized in each of thetopological forms characteristic of PrP (FIG. 1B).

[0094] Plasmid Constructions

[0095] Standard techniques were used in the creation of all plasmidconstructs (Sambrook et al 1989). All constructs were made in the pSP64vector (Promega, Madison, Wis.) containing the 5′ UTR of Xenopus globininserted at the HindIII site. Prl-PrP was constructed by PCRamplification of the region encoding amino acids 1-30 of preprolactinand amino acids 23-28 of PrP. The PCR product was ligated into aBgl2-PflmI digested vector containing wild-type PrP (PrP SV12), so thatthe resulting clone contained a precise fusion of the preprolactinsignal sequence to the mature region of PrP. All other PrP, AV3, andG123P signal sequence replacements were constructed similarly, exceptthat for Prl-PrP_((AV3)), the site of fusion was engineered three aminoacids downstream of the βL signal cleavage site. This clone wasidentical in final topology to Prl-PrP_((AV3)) constructed as a perfectfusion. The μL signal sequence was found to contain an Asp at position 2rather than Ser. This replacement had no significant effect on βL-PrPtopology. PrP_((R2, 3)); PrP_((R4, 5)); PrP_((D2, 3)); and PrP_((D4, 5))were created by first introducing a silent NheI site at codon 8 of PrPSV12, and then ligating annealed oligos encoding these mutations asBgl2-NheI fragments. To fuse these signals sequences to the prolactinmature region, the latter (beginning at amino acid 34) was PCR-amplifiedand digested with NcoI. This fragment was ligated into the wild-typepreprolactin vector (pSP BPI) digested at NcoI, effectively deleting thefirst 33 amino acids of prolactin. An XbaI site, encoding Thr-Arg, wasengineered immediately upstream of amino acid 34, and PrP, preprolactin,pre-beta-lactamase, and prepro-alpha factor signal sequences wereamplified and inserted as HindIII-SpeI fragments. Prl-βL was constructedusing an identical scheme. All clones were verified by dideoxysequencing.

[0096] Cell-Free Translation and Proteolysis

[0097] In vitro transcription with SP6 RNA polymerase, translation withRRL, and translocation into canine rough microsomes have been described(Hegde et al, (1998) Cell 92, 621-631 and references therein).Translations were carried out at 32° (or 26° in FIG. 4C) for 20-45minutes. Where indicated membranes were isolated by sedimentation andresuspended in physiologic salt buffer (PBS) as described (Hegde andLingappa, (1996) Cell 85, 217-228). Proteolysis with 0.5 mg/ml PK wasfor 45-90 minutes at 0°. Reactions were terminated with 12.5 mM PMSF andtransferred into 10 volumes of 1% SDS at 100°.

[0098] Targeting Studies

[0099] In FIG. 2B, translation in the absence of membranes was initiatedat 32°, following a 30 second pre-incubation in the absence oftranscript. Five minutes after transfer to 32°, aurintricarboxylic acid(ATA—Sigma) was added to 75 μM. Aliquots were removed to ice atstaggered intervals. Upon collection of the last sample, membranes wereadded to all but one aliquot. The samples were then returned to 32° for30 minutes, followed by proteolysis and immunoprecipitation with 3F4. InFIG. 2C, PrP truncated at StuI was translated at 32° in the presence orabsence of microsomes. Five minutes into this incubation, ATA was addedas above. After 30 minutes of translation, samples were removed to ice,membranes were added to the samples lacking them, and incubation at 32°was continued for 30 minutes. After translation was completed, chainswere released with 10 mM EDTA at 26° for 10 minutes. Proteolysis andimmunoprecipitation followed.

[0100] Cross-Linking

[0101] For samples to be cross-linked, translation products weresedimented and resuspended as above, and divided into equal aliquots.One aliquot was set aside, and to the other DSS was added to 1 mM andthe sample was incubated at room temperature for 30 minutes. Reactionswere terminated by the addition of 50 mM Tris (pH 8.0), 10 mM EDTA, and10 μg/ml RNase A (Sigma, St. Louis, Mo.). Where the isolation of lumenalcross-links was desired, 0.5% saponin was included as well, followed bysedimentation for 10 minutes at 75,000 rpm, 4°, in a TLA100. Forimmunoprecipitation of cross-linked material, saponin was added asabove, and antibody was added directly to the quenched cross-linkingreaction.

[0102] Microsomal Membrane Fractionation.

[0103] Briefly, rough microsomal membranes (RMs) are prepared aspreviously described (Walter and Blobel Methods Enzymol (1983)96:84-93). Following the extraction of lumenal and peripheral membraneproteins, a subset of the integral membrane proteins are solubilizedusing detergent and fractionated by a combination of lectin affinity andion-exchange chromatography. Individual fractions are reconstituted byremoval of detergent in the presence of lipids, and the proteoliposomesthat form are collected and used to assay for substrate-specificactivities. The following is the detailed protocol for the preparationand characterization of an initial set of fractions that demonstrate theprinciples involved. This initial procedure may be modified in severalways as detailed in section [d] below.

[0104] Fractionation protocol: i) RMs are prepared according topreviously published protocols (Walter and Blobel (1983) supra).Briefly, pancreatic tissue from a recently deceased dog (or pig) ishomogenized, and after a centrifugation step to remove debris, nucleiand large subcellular structures, the remaining material is subject tohigh-speed centrifugation. Sedimented material is resuspended and storedfrozen at −80° C. All subsequent procedures are carried out either onice or in a cold room at 4° C., unless otherwise noted. ii) The RMs (in50 mM triethanolamine-acetate, pH 7.4, 250 mM sucrose, 1 mM DTT) arediluted to a final concentration of 0.5 equivalents per μl with a buffercontaining 50 mM Hepes, pH 7.4, 250 mM sucrose. Saponin is added to afinal concentration of 0.5% from a 20% stock solution. After thesolution is mixed gently, but thoroughly, the microsomes are isolated bycentrifugation at 100,000 rpm for 10 minutes in a TL100.3 rotor (Beckmaninstruments). The pellet is resuspended at a concentration of 0.5equivalents per μl in buffer containing 500 mM K-acetate, 50 mM Hepes,10 mM EDTA, 125 mM sucrose. The microsomes are again isolated bycentrifugation (100,000 rpm for 20 min in TL100.3 rotor) and resuspendedat 1 equivalent per μl in extraction buffer [350 mM K-acetate, 50 mMHepes, pH 7.4, 5 mM MgCl₂, 15% w/v glycerol, 5 mM 2-mercaptoethanol,EDTA-free protease inhibitors (Roche Molecular Biochemicals) and 0.8%deoxy-BigCHAP (Calbiochem)]. Following extraction for 10 minutes on ice,insoluble material is sedimented at 100,000 rpm for 30 minutes in theTL100.3 rotor. The supernatant is termed the DBC extract, and used insubsequent steps. iii) The DBC extract is incubated with one-fifth toone-seventh volume of immobilized Con A (Pharmacia) with constant andgentle mixing for between 10 and 15 hours, at 4° C. The supernatant (ConA-depleted DBC extract) is removed to a separate tube and either savedon ice (for up to 12 hrs) or for extended storage, frozen in liquidnitrogen and kept at −80° C. The Con A beads are washed three times infive to seven volumes of wash buffer [500 mM K-acetate, 50 mM Hepes, pH7.4, 5 mM MgCl2, 15% w/v glycerol, and 0.5% deoxy-BigCHAP], and thebound proteins eluted with 5 volumes of elution buffer [500 mMK-acetate, 50 mM Hepes, pH 7.4, 20 mM EDTA, 15% w/v glycerol, 250 mMmethyl-alpha-D-mannopyrannoside, 2 mM 2-mercaptoethanol and 0.5%deoxy-BigCHAP] by incubation for 2 hours at 25° C. with constant mixing.The eluted material (Con A elutate) is chilled on ice for subsequentmanipulations. iv) The Con A eluate is diluted with 1.5 volumes ofdilution buffer [50 mM Hepes, pH 7.4, 15% w/v glycerol] and divided forincubation with ion-exchange resins. The diluted Con A eluate is addedto one-twentieth volume of either Q-sepharose-Fast Flow orS-sepharose-Fast Flow (both from Pharmacia), and incubated for 1 hourwith constant mixing. The unbound fraction of the Q-sepharose-Fast Flowand S-sepharose-Fast Flow incubations are added to one-twentieth volumeof either S-sepharose-Fast Flow or Q-sepharose-Fast Flow, respectively,and incubated for one hour at 4° C. with constant mixing. The unboundmaterial of this incubation is set aside (Q/S-flow thru and S/Q-flowthru). The resin from the second incubation (with S-sepharose andQ-sepharose) are washed in 10 volumes of buffer containing [200 mMK-acetate, 50 mM Hepes, pH 7.4, 15% w/v glycerol, 0.5% deoxy-BigCHAP],and subsequently eluted in 4 volumes of the same buffer containing 1000mM K-acetate. These fractions are termed the Q-FT and S-FT,respectively. The resin from the first ion-exchange incubations (withQ-sepharose and S-sepharose) are washed in 10 volumes of buffercontaining [200 mM K-acetate, 50 mM Hepes, pH 7.4, 15% w/v glycerol,0.5% deoxy-BigCHAP], and subsequently eluted in four volumes of the samebuffer containing 500 mM K-acetate. The resin is eluted a second time infour volumes of the same buffer containing 1000 mM K-acetate. Thesefractions are termed Q-500, S-500, Q-1000, and S-1000, respectively. v)The fractions are reconstituted as follows. First, lipids are preparedby mixing 8 mg phosphatidyl choline (PC, from a 10 mg/ml stocksolution), 2 mg phosphatidyl ethanolamine (PE from a 10 mg/ml stocksolution), 10 mg deoxy-BigCHAP (DBC, from a 100 mg/mil stock solution),and DTT added to 10 mM from a 1M stock. The sample is dried under vacuum(ne heat) and resuspended in 500 μl of buffer [50 mM Hepes, pH 7.4, 15%glycerol]. DBC is added from a 100 mg/ml stock solution to bring thefinal concentraion to approx. 20 mg/ml (˜2% w/v). The lipid mixture isfrozen in liquid nitrogen and stored at −80° C. until needed. Forreconstitutions, 100 μl of the DBC extract or ConA depleted DBC extractis mixed with 100 μl of either Con A elution buffer, Con A eluate, S-FT,Q-FT, S-500, Q-500, S-1000, or Q-1000 fractions prepared as describedabove. In addition, 5 μl of the lipid mixture is added and the samplemixed thoroughly before adding the individual samples to 160 mg ofBioBeads SM2 (Biorad). The mixtures are incubated for 12-18 hours at 4°C. with constant mixing. vi) To recover the proteolipososmes, the liquidphases from step (v) are separated from the biobeads and transferred tofresh tubes. These samples are diluted with five volumes of ice-colddistilled water, mixed, and transferred to 1.3 ml centrifuge tubes onice. The samples are centrifuged for 20 min in TL100.3 rotor (withadaptors for the 1.3 ml tubes) at 70,000 rpm. The supernatant isdiscarded and the pelleted proteoliposomes are resuspended in 30 μl of abuffer containing 100 mM K-Acetate, 50 mM Hepes, pH 7.4, 250 mM sucrose.These are frozen in liquid nitrogen and stored at −80 until used inassays for translocation. vii) Translocation assays are performed asdescribed previously (ref.) using 1 μl of the proteoliposomes from step(vi) per 10 μl of translation reaction.

[0105] Purification of PDIp

[0106] 20,000 equivalents (see Walter and Blobel, 1983, for definition)of canine pancreatic rough microsomal membranes were adjusted to 80 mlsin 50 mM triethanolamine, 250 mM sucrose, 0.2 mM PMSF, 5 μg/mlaprotinin, 10 μg/ml chymostatin, 1 μg/ml E64, 5 μg/ml antipain, 1 mMDTT. With constant mixing, purified saponin was added slowly to 1% w/vfinal concentration, and after incubation at 4° C. for 15 minutes,membranes were sedimented by centrifugation for 2 h at 70,000 rpm in70.1 Ti rotor (Beckman). The supernatants were pooled and applied to a 5ml column of ConA sepharose at a flow rate of 6.2 mls/hr. The column waswashed at 15 ml/hr with 25 mls of the above buffer containing 100 mMKAc, and an additional 25 mls with the above buffer without KAc. Thecolumn was eluted at 3 ml/hr at room temperature with 20 mls of theabove buffer containing 1 M α-methyl-mannopyrannoside. The eluate wascollected on ice and applied at 4° C. to a 1 ml Q-sepharose fast flowcolumn. The column was washed with 4 ml of 50 mM Hepes, pH 7.5, 2 mMMgAc, 1 mM CaCl2, 1 mM DTT and eluted with 2 mls of this same buffercontaining 1 M KAc. The eluate was further fractionated on a Superdex PG16/16 column (Pharmacia) and 80 1.5 ml fractions collected. 15 μl eachof fractions 31-56 were analyzed by SDS-PAGE and coomassie staining(FIG. 4b). Peak fractions containing gp65 were pooled and aliquots usedfor subsequent sequence analysis (by ProSeq, Salem, Mass.).

[0107] Preparation of TRAM-Reconstituted Membranes

[0108] Glycoprotein-depleted membranes were prepared as described (Hegdeet al, 1998c). Purified TRAM, prepared as described (Gorlich andRapoport, 1993), was added at 4× the level present in starting membranes(as judged by immunoblotting) to the glycoprotein-depleted extract.

[0109] Miscellaneous

[0110] SDS-PAGE was performed using either 15% gels or 12.5% or 15%Tris/Tricine gels. Bands were either visualized directly byautoradiography, or with the aid of a TranScreen LE intensifying screen(Kodak; Rochester, N.Y.). Quantitation was performed by scanning on anAGFA ArcusII flatbed scanner and densitometry using Adobe Photoshopsoftware. Immunoprecipitations were as described (Chuck and Lingappa,1992)

[0111] While all three proteins were efficiently targeted andtranslocated across the ER, they differed dramatically in theirtopological outcomes. Consistent with previous findings, PrP wassynthesized predominantly in the ^(sec)PrP and ^(Ntm)PrP topologicalforms, with a small yet significant amount of ^(Ctm)PrP (˜7%; FIG. 1C).By contrast, Prl-PrP was synthesized predominantly in the ^(sec)PrPform, followed by lesser amounts of ^(Ntm)PrP, and essentiallyundetectable amounts of ^(Ctm)PrP (<2%). Conversely, the topology ofβL-PrP was dramatically shifted toward ^(Ctm)Prp (˜34%) largely at theexpense of ^(sec)PrP. These results suggest that different signalsequences encode information which dramatically affects subsequenttopological events. This information appears to be in addition to anddependent on the basic targeting feature common to all three signalsequences. Thus, a chimeric protein consisting of the N-terminal twentyamino acids of the cytosolic protein globin fused to mature PrP fails totranslocate in any of the topological forms (data not shown). Takentogether, the experiments in FIG. 1 demonstrate that the signal sequenceof PrP plays a role in topological regulation.

[0112] In principle, the influence of the signal sequence on topologydescribed above could be ascribed to the well established role of signalsequences in targeting. If the different signal sequences were to targetto the ER at different rates, then the N-terminus of the maturesubstrate would be synthesized to varying lengths by the time theribosome-nascent chain complex interacts with the translocon. Oneconsequence of increasing amounts of synthesis during the targeting stepmay be to favor synthesis of one of the topological forms of PrP (mostlikely ^(Ctm)Prp, the N-terminal domain of which is not translocated).This hypothesis implies that manipulation of the kinetics of PrPtargeting should recapitulate the shifts in topology seen byreplacements of the signal sequence. This possibility was tested inthree ways.

[0113] First, the amount of ER-derived canine pancreatic microsomespresent in the translation reactions was titrated and the effect on PrPtopology was examined. As the total concentration of the translocationmachinery decreases, the time between initiation of synthesis andinteraction with the translocon should increase, allowing increasingamounts of the nascent chain to be synthesized prior to targeting. Wefound that varying the concentration of microsomes over a 10-fold rangedid not impact the relative ratios of the different topological forms ofPrP (FIG. 2A). The decrease in translocation efficiency with decreasingmicrosome concentration, reflected by a decrease in efficiency of signalcleavage, is likely due to the loss of “translocational competence,” therelatively brief period in nascent chain growth when theribosome-nascent chain complex is able to interact productively with thetranslocation machinery (Perara et al. Science (1986) 232:348-352). Asimilar loss of translocation efficiency was observed with othersubstrates (data not shown).

[0114] In a second approach, translation reactions were synchronized byinhibiting initiation after 5 min, and microsomes were added atsuccessively later times. We found that regardless of the duration forwhich translation was allowed to occur prior to the addition ofmicrosomes, the percentage of ^(Ctm)PrP synthesized remained constant(FIG. 2B). Again the efficiency of signal cleavage decreased at thelater time points due to an increasing percentage of nascent chains thathad presumably lost translocational competence.

[0115] Finally, the targeting step itself was synchronized. In theseexperiments, PrP mRNA was truncated at a defined length and translatedin the absence of microsomal membranes. The resultingribosome-associated nascent chains remain competent for translocation inthe absence of further translation, and can be targeted synchronously bysubsequent addition of microsomes (Perara et al., 1986, supra).Following targeting, the ribosomes were disassembled, and the topologyachieved by the truncated PrP chain, which lacks only a small portion ofthe PrP C-terminus, was compared to the same truncated product that wastargeted co-translationally. We found that synchronizing the targetingof this 223-mer of PrP did not affect the relative ratios of thetopological forms observed (FIG. 2C). Similar results were obtained fora 180-mer as well (data not shown). Taken together, these experimentscollectively argue that the manipulation of the kinetics of thetargeting step of PrP is insufficient to alter the final topology thatis achieved. We infer from these findings that the topology-specificinformation encoded within a signal sequence is likely to act at a stepbeyond targeting.

[0116] Since differences in the kinetics of targeting cannot account forthe influence of the signal sequence on PrP topology, we turned to theother site of signal sequence action, the ER membrane. Signal sequencerecognition events at the ER membrane appear to occur early duringtranslocation and precede the establishment of a tight ribosome-membranejunction. If the preprolactin and pre-bata-lactamse signal sequencesencode innate differences in the mechanism of their recognition by thetranslocation machinery, these differences might be manifested in thestate of the junction.

[0117] Short translocation intermediates of preprolactin andpre-beta-lactamase were analyzed for their accessibility to proteinase K(PK), a probe for the state of the ribosome-membrane junction.Consistent with previous reports (Connolly et al., J. Cell Biol (1989)108:299-307; Jungnickel and Rapoport, Cell (1995) 82:261-270), the tightseal of the junction shielded the preprolactin 86-mer from proteolyticattack. By contrast, the pre-beta-lactamase 84-mer was largelysusceptible to digestion, indicative of an open ribosome-membranejunction (FIG. 3A). To determine if this difference was attributable tosignal sequence function, we exchanged the signal sequences ofpreprolactin and pre-beta-lactamase (constructs βL-Prl and Prl-βL). Wefound that the βL-Prl 81-mer was accessible to protease digestion whilethe Prl-βL 93-mer was not (FIG. 3A). Similar results were obtained withtranslocation intermediates approximately 20 amino acids longer (datanot shown). These data argue that signal sequences can differentiallyinfluence the state of the ribosome-membrane junction during the earlybiogenesis of secretory proteins.

[0118] Given the above results, modulation of the ribosome-membranejunction by the signal sequence seemed to be a plausible mechanism forthe regulation of PrP topology. If so, the topological form of PrP withits N-terminus in the cytosol (^(Ctm)PrP) may be expected to arise fromchains that did not establish a tight ribosome-membrane junction earlyin translocation. To test this hypothesis, increasingly longertranslocation intermediates of PrP, Prl-PrP, and βL-PrP were assembledat the ER and separated from non-targeted chains by sedimentation, andthe state of the ribosome-membrane junction was analyzed by PK digestion(FIG. 3B).

[0119] When truncated at a point corresponding to a 61-mer of wild-typePrP, all three substrates are fully accessible to protease, and thushave failed to establish a tight junction. However, the three constructsshow differential protease accessibility at a translocation intermediateonly 52 amino acids longer, even before the synthesis of thetransmembrane domain. Prl-PrP, which makes very little ^(Ctm)PrP, islargely shielded from digestion at this point, while a greaterpercentage of βL-PrP chains are accessible to the protease. At thistruncation point, all three constructs are resistant to extraction fromthe membrane with high salt (0.5 M potassium acetate; data not shown),suggesting that the ribosome is stably associated with the translocationchannel. Furthermore, βL-PrP nascent chains remain more accessible to PKthan Prl-PrP chains throughout their biogenesis. Most of the unprotectedchains retain their signal sequences, likely as a consequence of theopen junction. The observed diminishment of protease protection in allthree substrates with increasing chain length probably reflectsjunctional opening during the synthesis of ^(Ntm)PrP. Collectively,these results argue that signal sequence-mediated regulation of theribosome-membrane junction can have dramatic consequences for subsequenttranslocational events.

[0120] The putative role of the ribosome-membrane junction in governingfinal topology suggests that trans-acting factors which regulate thejunction could influence PrP biogenesis. The TRAM glycoprotein isthought to interact with signal sequences to facilitate the initialassociation between the ribosome-nascent chain complex and thetranslocon (Voigt et al., J. Cell Biol (1996) 134:25-35). The signalsequences of some substrates (such as preprolactin) do not depend onTRAM for translocation, though most (including pre-beta-lactamase) areTRAM-dependent (Gorlich et al., Nature (1992) 357:47-52; Gorlich andRapoport, Cell (1993) 75:615-630; Voigt et al., 1996, supra). AlthoughTRAM has additionally been implicated in regulating theribosome-membrane junction during the translocation of other substrates(Hegde et al., Cell (1998) 92:621-631), functional studies have thus farfailed to demonstrate a direct or critical role for TRAM in PrP topology(Hegde et al., Mol. Cell (1998) 2:85-91). Thus, we reasoned that itshould be possible to dissociate the TRAM-dependence feature of a signalsequence from its role in PrP topology.

[0121] We sought to identify mutations within the PrP signal sequencethat differentially modify topology but not dependence on TRAM. Byanalogy to the effects of charge on the topology of signal-anchorproteins (e.g., Sipos and von Heijne, Eur J. Biochem (1993)213:1333-1340; Spiess, FEBS Lett (1995) 369:76-79), we reasoned thatnon-conservative mutations in the PrP signal sequence may affect PrPbiogenesis. Leaving the hydrophobic core of the signal sequence intactto allow efficient targeting, we replaced either codons 2 and 3 orcodons 4 and 5 with arginine or aspartic acid codons [PrP_((R2, 3)),PrP_((D2, 3)), PrP_((R4, 5))and PrP_((D4, 5)), respectively; see FIG.4A). Topological analysis of PrP_((R2, 3)) and PrP_((R4, 5)) revealed adecrease in ^(Ctm)PrP synthesis while PrP_((D2, 3)) and PrP_((D4, 5))both showed increased ^(Ctm)PrP synthesis (FIG. 4B).

[0122] To assess the TRAM dependence of these mutant signal sequences wefused each of them to preprolactin in place of the native preprolactinsignal sequence. The ability of these constructs to translocate intoglycoprotein-depleted proteoliposomes either containing or lackingpurified TRAM was measured. TRAM-dependence was defined as thepercentage of chains that require TRAM to translocate. We found that allfour mutated signal sequences were less TRAM-dependent than thewild-type PrP signal sequence, despite their divergent effects on PrPtopology (FIG. 4C). Thus, the TRAM-dependence of the PrP signal sequencecan be dissociated from the role of the signal sequence in regulatingtopology, arguing for a previously unappreciated mechanism by whichsignal sequences influence topology.

[0123] Mutations in the membrane-spanning domain can influence thetopology of PrP (Hegde et al., Science (1998) 279:827-834). Becausetransmembrane (TM) domains, like signal sequences, can elicit changes inthe ribosome-membrane junction (Liao et al., Cell (1997) 90:31-41), itis plausible that the mechanisms by which these two domains regulatetopology are related. Just as certain signal sequences open theribosome-membrane junction, the TM domain of PrP may be capable ofeliciting an opening of the junction. In this scenario, TM domainmutants that favor ^(Ctm)PrP would elicit a greater opening of thejunction than wild-type PrP, while mutants that favor ^(sec)PrP wouldact to maintain a closed junction. We tested this hypothesis byanalyzing PrP constructs that contained various combinations of changesin both the signal sequence and TM domains.

[0124] Replacement of conserved alanines in the TM domain with valinesmarkedly increases the proportion of PrP made in the ^(Ctm)PrP topology(Hegde et al., Science (1998) 279:827-834). One of these mutants[PrP_((Av3)), which generates approximately 40% of the molecules in the^(Ctm)PrP form] was engineered with either a preprolactin orpre-beta-lactamase signal sequence [Prl-PrP_((Av3)) and βL-PrP_((Av3))].If the AV3 mutation causes the ribosome-membrane junction to openindependently of the signal sequence, then the Prl-PrP_((AV3)) constructshould synthesize roughly as much ^(Ctm)PrP as PrP_((Av3)). Strikingly,we observed that Prl-PrP_((Av3)) reduces ^(Ctm)PrP synthesis to barelydetectable levels (FIG. 5A, middle panel). Conversely, βL-PrP_((Av3))makes almost exclusively ^(Ctm)PrP (FIG. 5A, right panel).

[0125] Similarly, according to the hypothesis stated above, a TM mutantwhich favors ^(sec)PrP synthesis should cause the ribosome-membranejunction to close. When we replaced the signal sequence of the TM mutantPrP_((G123P)), which makes only ^(sec)PrP with the preprolactin orpre-beta-lactamase signal sequence [Prl-PrP_((G123P)) andβL-PrP_((G123P))], we found that all three substrates were synthesizedonly as ^(sec)PrP However, closure of the ribosome-membrane junctionshould stimulate overall translocation efficiency, since chainssynthesized with a closed junction have the ER lumen as their onlyoption for exit from the ribosome. Instead, we found thatβL-PrP_((G123P)) translocates relatively poorly compared toPrP_((G123P)) and Prl-PrP_((G123P)). This result argues that the G123Pmutation does not necessitate the production of ^(sec)PrP by closing theribosome-membrane junction, but rather by preventing the translocationof chains which would otherwise become ^(Ctm)PrP.

[0126] The findings above suggested that rather than actingindependently of the signal sequence, the TM domain is constrained bythe preceding action of the signal sequence. To test directly whetherthe TM domain has any bearing on the state of the ribosome-membranejunction dictated by the signal sequence, we analyzed the state of thejunction for Prl-PrP_((AV3)) and βL-PrP_((G123P)) at a point after theemergence of the TM domain. We found that the junction remains open forβL-PrP_((G123P))and closed for PrI-PrP_((Av3)), irrespective of theidentity of the TM domain (FIG. 5C). These results suggest that theinitial action of the signal sequence on the ribosome-membrane junction,even before the synthesis of the TM domain, is critical to subsequenttopological determination.

[0127] The observation that PrP chains are committed to a transmembranetopology prior to the synthesis of the TM domain was unexpected. Whymight it be important to place the key regulatory steps at such an earlypoint in biogenesis? One possible reason is that certain early events inthe folding of PrP are not readily reversible at a later point inbiogenesis when the TM domain has been synthesized. Thus, it may beimportant to initiate these early folding events in a temporally andspatially restricted manner (given that the N-terminus can ultimatelyreside in one of two environmentally different compartments). To examinethis possibility, we sought to identify interactions between PrP and themachinery of protein folding that are initiated in a topology specificmanner at a time before topology has been formalized.

[0128] In initial experiments, we used cross-linking to identifyproteins that displayed preferential association with eitherPrP_((G123P))or PrP_((AV3)). While several proteins interact comparablywith both substrates, a 65 kDa protein (p65) cross-links more stronglyto PrP_((G123P))than to PrP_((AV3)) (FIG. 6A). We then similarlyanalyzed Prl-PrP and βL-PrP truncated at the same location and foundthat p65 preferentially cross-links to Prl-PrP, suggesting that nascentPrP chains in the process of being made in the ^(sec)PrP topologyspecifically associate with this factor (FIG. 6B). To determine whenduring PrP biogenesis this interaction is initiated, we examined earlytranslocation intermediates of Prl-PrP and βL-PrP for the differentialpresence of this cross-link. Remarkably, we found that p65 and aslightly smaller protein cross-link preferentially to the Prl-PrPtranslocation intermediate truncated at codon 113 of wild-type PrP (FIG.6C). Thus, association of p65 with nascent PrP chains very early inbiogenesis correlates with the propensity of the substrate to ultimatelybe made in the ^(sec)PrP topology.

[0129] We took advantage of the biochemical properties of the p65cross-linked adduct to purify and identify this protein. The adduct wasextractable by saponin, implying a lumenal localization for p65; itmigrated as a 4 S protein by sucrose gradient analysis, suggesting itwas monomeric; it was retained on a ConA-sepharose column, indicatingthat p65 is glycosylated; and it was also retained on a Q-sepharosecolumn, suggestive of a negative net charge imparted by p65 on anotherwise net-positive PrP molecule. When these properties were combinedin a stepwise fractionation of canine rough microsomes, a single major65 kDa protein was purified (FIG. 6D). Sequencing of peptide fragmentsgenerated by cyanogen bromide cleavage of this protein identified it asPDIp, a pancreas-specific member of the Protein Disulfide Isomerase(PDI) family of chaperones (Desilva et al., Cell Biol (1996) 15:9-16;Elliott et al., Eur J. Biochem (1998) 252:372-377).

[0130] The identity of p65 as PDIp was confirmed by immunoprecipitationwith antibodies raised against PDIp (FIG. 6E). In addition, the slightlysmaller cross-linking partner was immunoprecipitated by antibodies toubiquitous PDI, further arguing for a specific interaction between PrPand this family of proteins. The immunoprecipitation studies confirmedthat both PDI and PDIp cross-link more strongly to Prl-PrP than toβL-PrP (FIG. 6E). This differential crosslinking of ^(sec)PrP favoringconstructs to PDI was also observed in microsomes isolated from brain,the tissue in which PrP is predominantly expressed. In this case, onlycross-links to the ubiquitous PDI were observed, consistent withprevious observations that PDIp is not expressed strongly in braintissue (Desilva et al., 1996, supra).

[0131] We next sought to determine if ^(sec)PrP specifically interactswith any other lumenal proteins early during biogenesis. Examination ofthe saponin extractable cross-links revealed prominent interactionsbetween the ^(sec)PrP favoring constructs [PrP_((R2, 3)) andPrP_((R4,5))] and proteins of approximately 30 kDa, 60 kDa, and 65 kDa(FIG. 6F). Similar cross-links to the ^(Ctm)PrP favoring mutants[PrP_((D2, 3)) and PrP_((D4, 5))] were markedly diminished. As expected,the 60 kDa and 65 kDa cross-links were identified by immunoprecipitationas PDI and PDIp (data not shown). The identity of the 30 kDacross-linking partner remains unknown. These cross-links are notobserved simply as a fortuitous consequence of the lumenal localizationof ^(sec)PrP because prominent cross-links to lumenal proteins were notobserved with early translocation intermediates of preprolactin (FIG. 6Fand data not shown). The cross-linking data collectively argue that oneconsequence of signal sequence-mediated events in early PrP biogenesisis to facilitate differential interactions between the nascent chain andfactors which may subsequently participate in its biogenesis.

Example 2 Pathogensis of Altered Topology of Prion Protein is Common toGenetic and Infections Prion Diseases

[0132] Prion diseases can be infectious, sporadic and genetic (Prusiner,S. B. PNAS. (1998) 95:13363-13383; Weissmann, C J.Biol. Chem (1999)274:3-6; Johnson New Eng J Med (1998) 339:1994-2004; Horwich Cell (1997)89:499-510). The infectious forms of these diseases, including bovinespongiform encephalopathy and Creutzfeldt-Jakob disease, are usuallycharacterized by the accumulation in brain of the transmissiblepathogen, and abnormally folded insoform of the prion protein (PrP)termed PrP^(Sc.). However, certain inherited PrP mutations appear tocause neurodegeneration in the absence of PrP^(Sc) (Brown, Ann Neur(1994) 35:513-529; Tateishi, J. et al., Neurology, 1990, 40:1578-1581;Tateishi, Neurology, (1996) 46:532-537, Tateishi, J., Brain Pathology,(1995) 5:53-59), instead working by favoured systhesis of ^(Ctm)PrP, atransmembrane form of PrP (Hegde, et al. Science (1998) 279:827-834).Certain mutations in PrP (including the human prion disease-associatedA117v mutation) alter its biogensis at the endoplasmic reticulum (ER),causing a higher percentage of PrP molecues to be synthesized in thetransmembrane form ^(Ctm)PrP. Expression of ^(Ctm)PrP-favouringmutations in transgenic mice resulted in neurodegenerative changessimilar to those observed in prion disease (Hegde, et al. Science (1998)279:827-834) The detection of ^(Ctm)PrP-favouring mutations suggestedtheat elevated ^(Ctm)Prp causes neurodegeneration (Hegde, et al. Science(1998) 279:827-834), but whether this mechanism of neurodegeneration isinvolved in the pathogenesis of transmissible prion disease has beenunclear. To explore this question mice were generated with mutant PrPtransgenes differing in their propensity to form ^(Ctm)PrP, andsubsequently their suceptibility to PrP^(Sc) induced neurodegenerationwas assessed.

[0133] Cell-free translation and translocation. Transcription of therelevant coding regions using SP6 polymerase, translation in rabbitreticulocyte lysate containing imcrosomal membranes from dog pancreas,and proteolysis were essentially as described previously (Hegde, et al.Science (1998) 279:827-834). Translation reactions were carried out at32° C. for 40 minutes, and proteolysis reactions at 0° C. for 60 minutesusing 0.5 mg/ml PK. Products were immunoprecipitated with the R073antibody (Rogers, M. et al., J. Immun, 1991, 147:3568-3574), separatedby SDS-PAGE on 15% acrylamide gels, and visualized by autoradiography.TABLE 1 Transgenic mouse production and characterization. Level of PrPAge spont Transgenic Transgenic % Ctm in expr (rel to disease CtmPrP inSc237 inc. Line Number Line Name vitro Sha) Ctm-index (days) vivo time(days) TgSHaPrp(S F1788  6 4  24 − − 323 +/− 14 TE)H (9/9) TgSHaPrPE15781 31 0.4  12 − −  70 +/− 2 (A117V)L (6/6) TgSHaPrP E15727 31 4 124572 +/− 35 +  55 +/− (6/6) (A117V)H (5/5) (6/6) TgSHaPrP(N E15786 35 1 35 − − 311 +/− (3-3) 1081)L TgShaPrp(N E15790 35 5 175 312 +/− 24 + 233+/− 2 1081)H (7/7) (9/9) TgShaPrP(K E12485 48 0.4  19 − − 257 +/− 2H-II)L (9/9) TgSHaPrP(K F1220 48 1  48 472 +/− 13 + 181 +/− 5 H-II)M(6/6) (10/10) TgSHaPrP(K F1198 48 4 192  58 +/− 11 ++ ND^(b) H-II)H(24/24)^(a)

[0134] Table 1 Characteristics of transgenic mouse lines used in thisstudy. The values for % Ctm in vitro were derived from quantitation ofFIG. 8a. The levels of PrP expression were determined by quantitivewestern blotting with the 13A5 monoclonal antibody and re rexpressedrelative to PrP expression in Syrian hamster *Sha; see FIG. 1B for arepresentative experiment). The Ctm-index for each transgenic line isderived by multiplying the values in the preceeding two columns. ‘Agespont disease’ indicates the age of onset of clinical symptons[average+/−SEM (n/n.)]. biochemical assay for determining the presenceof ^(Ctm)Prp in vivo was as previously described^(9,) and carried out oneither clincially ill animals (in the case of transgenic linesdeveloping illness) or mice over 600 days of age (in the case oftransgenic lines that don't develop neurodegeneration). ‘Sc237 inc.time’ indicates the time from inoculation with Sc237 Sha prions todevelopment of neurologic signs of dysfunction [average+/−SEM (n/n.)].were generated as previously described (Manson, et al. Neurodegeneration(1994) 3:331-340 and references therein). PrP expression was assessed byimmunoblotting of brain tissue homogenate with 13A5 mAb (Kascsak,R. J.et al., J. Virology, 61:3688-3693), comparing to serial dilutions ofnormal Syrian hamster brain tissue (FIG. 8b and Table 1). Observation ofthese mice for development of spontaneous illness was as previouslydescribed (Prusiner Ann Neur (1982) 11:353-358). The double trangenicmice expressing both SHaPrP and MoPrP (see FIG. 11) were generated bycrossing Tg[SHaPrP]/Prnp^(0/0) (line A3922)⁹ to Tg [MoPrP]/Prn^(0/0)(line B4053) (Telling Genes Dev (1996) 10:1736-1750). Transmissibility(see FIG. 10) was assessed by intracerebral inoculation of 1% brainhomogenate (w/v) into mice (30 μl per animal) or hamsters (50 μl peranimal) as previously described (Prusiner Ann Neur (1982) 11:353-358).the Sc237 strain of hamster prions (used in FIG. 9) and RML strain ofmouse prions (used in FIG. 11) have been described previously (Marsh JInfect Dis (1975) 131:104-110; Chandler Lancet (1961) 1:1378-1379).

[0135] Assessment of brain for ^(Ctm)Prp and PrP^(Sc.) Brain tissue(either freshly removed or stored frozen at −80° C. followingflash-freezing in liquid nitrogen) was homogenized in PBS (at 5% w/v or10% w/v) by successive passage through 16, 18 and 20 gauge needles. For^(Ctm)PrP detection (‘mild’ proteolysis conditions), 17 μl aliquots ofthe sample (at a concentration of 25 μg/μl) were adjusted (in a finalvolume of 20 μl) to 1% NP-40, 0.25 mg/mk PK and incubated for 60 min onice. For PrP^(Sc) detection (‘harsh’ proteolysis conditions), 17 μlsamples (at a concentration of 25 μg/μl) were adjusted (in a finalvolume of 20 μl) to 0.5% NP-40, 0.5% deoxycholate, 0.1 mg/ml PK andincubated for 60 minutes at 37° C. It should be noted that thedifference between mild versus harsh digestion conditions, whileoperational, is not subtle, as it involves a 37° change in temperatureof incubation, and the presence of non-ionic detergent versus mixedmicelles of non-ionic and ionic detergents. The proteolysis reactionswere terminated by the addition of PMSF to 5 mM, incubating anadditional 5 minutes, and transferring the sample to 5 volumes ofboiling 1% SDS, 0.1M Tris, pH 8.9. Samples were then digested withPNGase as directed by the manufacturer, resolved by 10%tricine-SDS-PAGE, transferred to introcellulose, and probed with eitherthe 3F4 or 13A5 monoclonal anitbody (Kascsak J Virol 61:3688-3693), orthe RO73 polyclonal anitbody (Rogers J Immunol (1991) 147:3568-3574).

[0136] Results

[0137] Shown in FIG. 8a are in vitro translocation products of fourmutants of Syrian hamster (Sha) PrP that alter the amount of ^(Ctm)PrPsynthesized at the ER. Transgenic mice expressing each of these mutantPrPs in the FVB/Prnp^(0/0) background were generated and characterized(see Table 1 and FIG. 8b). The Tg[SHaPrP(KH→II)_(H)],Tg[SHaPrP(KH→II_(M)], Tg[SHaPrP(A117V)_(H)] and Tg{SHaPrP(N108I)_(H)]mice were observed to develop signs and symptons of neurodegenerativedisease at approximately 60, 472, 572 and 312 days, respectively (FIG.8c and Table 1). By contrast, neither the Tg[SHaPrP(ΔSTE)] mice nor miceexpressing lower levels of the disease-associated transgenes{Tg[SHaPrP(KH→II)_(L)], Tg[SHaPrP(A117V)_(L)] and Tg[SHaPrP(N108I)_(L)]}developed spontaneous disease (Table 1 and data not shown). biochemicalanalyses of brain tissue from each of these lines of transgenic micerevealed elevated ^(Ctm)PrP, but not PrP^(Sc), in the lines whichdeveloped disease (FIG. 8d). Together, the data in FIG. 8 recapitulatethe point that increased synthesis of the ^(Ctm)PrP form of PrP isassociated with the development of neurodegenerative disease.

[0138] More remarkable is the apparent dose response, seen in two ways,between ^(Ctm)PrP and severity of disease. First, the more strongly that^(Ctm)PrP systhesis is favoured at the ER (KH→II>N108I >A117V), theearlier the onset of spontaneous disease (Table 1). Second, lowering thelevel of expression of each of these mutations below an apparentthreshold abrogates both the generation of ^(Ctm)PrP (FIG. 8d and ref.9)and development of disease (Table 1). Furthermore, the three transgeniclines expressing the KH→II mutation develop disease at times inverselycorrelated with their respective levels of expression (Table 1). Theseobservations demonstrate that bot the ^(Ctm)PrP-favouring quality of amutation and its level of expression contribute to the development ofneuodegeneration.

[0139] This panel of transgenic mice with differing propensities to make^(Ctm)PrP was used to dissect the relationship between ^(Ctm)PrP andPrP^(Sc.). We first examined the susceptibility to PrP^(Sc) oftransgenic mice with identical levels of transgene expression butdiffering propensities to make ^(Ctm)PrP: Tg[SHaPrP(□STE)] andTg[SHaPrP(A117V)_(H)]. Upon inoculation with Sc237 hamster prions, wefound that the Tg[SHaPrP(□STE)] and Tg[SHaPrP(A117V)_(H)] mice developedillness at aproximately 323 and 54 days, respectively (FIG. 9a, Table1). Biochemical analysis of representative mice at the time of diseaseonset revealed that the Tg[SHaPrP(□STE)] mice contained substantiallymore PrP^(Sc) than the Tg[SHaPrP(A117V)_(H)] mice (FIG. 9b). Thus, thetransgenic line that generates higher CtmPrP is more susceptible toPrP^(Sc), developing disease at a lower level of overall PrP^(Sc)accumulation.

[0140] We next compared the susceptibility to Sc237 ofTg[SHaPrP(KH→II)_(L)] versus Tg[SHaPrP(A117V)_(H)] (FIG. 9e). By keepingthe mutation constant, issues regarding a potential barrier topropagation are avoided, while still changing the propensity to generateCtmPrP by modulating level of expression. As expected, lowering thelevel of expression increased the incubation time to disease following5c237 inoculation. More remarkably however, we found that with both theKH→II and A117V mutants, the lower level expressor contained the higherlevel of PrPSC at the time of disease onset (FIG. 9d, f). A similarinverse relationship between level of expression and amount of PrPSC atdisease has been observed with mice expressing different levels of wildtype PrP (ref. 10). Thus, as above, the mice with a diminishedpropensity to form OtmPrP had accumulated higher levels of PrPSC atonset of disease. To integrate the above inoculation data into a singleplot, we ranked the different lines of transgenic mice by their relativepropensities to generate CtmPrP using a measure we term the Ctm-index(see Table 1). This index is derived by multiplying the percent ofchains synthesized in the CtmPrP topology by the level of transgeneexpression, thus incorporating the two parameters known to influenceCtmPrP generation. FIG. 9g shows that a clear relationship existsbetween the Ctm-index and the amount of PrPSC that had accumulated atdisease onset. This relationship suggests that the ability of PrPSC tocause disease is a function of the propensity of the host to generateCtmPrP.

[0141] The data in FIG. 9 demonstrate two important points. First, verydifferent levels of PrP^(SC) accumulation are observed at the time ofonset of clinical disease upon inoculation of the various lines oftransgenic mice. Given that the strain of mice is identical in eachcase, with the only differences being in the nature and level ofexpression of the PrP transgene, this observation underscores theconclusion that accumulation of protease-resistant PrP^(Sc) is notlikely to be the most proximate cause of disease; subsequent events(apparently involving ^(Ctm)PrP) are likely to be involved. Second,there is a relationship between the amount of accumulated PrP^(SC) andthe factors that modulate ^(Ctm)PrP generation. Such a relationshipargues that ^(Ctm)PrP and PrP^(SC) are part of a pathway in which theypotentially can influence, either directly or indirectly, each other'smetabolism.

[0142] One way to reconcile the data in FIG. 9g is if accumulation ofPrP^(SC) caused increased generation of ^(Ctm)PrP which then elicitedneurodegeneration. Thus, transgenic mice that have an elevatedpropensity to generate ^(Ctm)PrP (i.e., a high Ctm-index) would requireless PrP^(SC) accumulation before ^(Ctm)PrP generation is increasedbeyond the threshold needed to cause disease. This model would explainthe inverse relationship observed in FIG. 9g, and makes two additionalpredictions. First, since transgenic mice that substantially favoursynthesis of PrP in the ^(Ctm)PrP form can entirely circumvent therequirement for PrP^(SC) in the developement of neurodegenerativedisease, tissue from such mice should not be infectious. Second,^(Ctm)PrP levels should increase during the course of PrP^(SC)accumulation in infectious prion disease. These predictions were tested.

[0143] To assess the transmissiblity of ^(Ctm)PrP-associated disease,brain homogenate from clinically ill Tg[SHaPrP(KH→II)_(H)] mice wereinoculated intracerebrally into four hosts: i) Tg[SHaPrP(KH→II)_(L)]mice expressing the KH→II mutation at low levels, ii) Tg[SHaPrP] miceoverexpressing wild type SHaPrP, iii) FVB/Prnp^(0/0) mice with ahomozygous disruption of the PrP gene, and iv) Syrian hamsters. As shownin FIG. 10, homogenate from terminally sick Tg[SHaPrP(KH→II)_(H)] micedid not induce neurological illness at rates different than controlTg[SHaPrP] homogenate when directly compared in three independent hosts.Furthermore, biochemical and pathological examination of representativebrain tissue from FIG. 10 at up to 625 days after inoculation did notshow any evidence of PrP^(Sc) or neurologic disease in eitherexperimental or control animals (data not shown). Yet, inoculation ofTg[SHaPrP(KH→II)_(L)] mice with Sc237 prions readily generated PrP^(Sc)in brain (FIG. 2d), which re-transmitted disease toTg[SHaPrP(KH→II)_(L)] and Tg[SHaPrP] mice (data not shown). Thus,although PrP(KH→II) is capable of being formed into PrP^(SC), the^(Ctm)PrP,. associated disease in Tg[SHaPrP(KH→II)_(H)] mice does notgenerate detectable PrP^(SC) and therefore is not infectious. Lack oftransmission provides further support for the hypothesis thatneurodegeneration in these genetic prion diseases is caused by ^(Ctm)PrPdirectly. The second prediction made on the basis of the data in FIG. 9gwas that accumulation of PrP^(SC) in infectious prion disease shouldinduce the increased generation of ^(Ctm)PrP, which would subsequentlylead to neurodegeneration. Unfortunately, testing this predictiondirectly is hampered by the biochemical properties of the accumulatingPrP^(SC) (Meyer PNAS (1986) 83:2310-2314). Being highlyprotease-resistant and heterogeneous in its fractionation, it tends tocontaminate substantially all subcellular fractions. Additionally, itinterferes with the assays for ^(Ctm)PrP detection, which is also basedon protection from proteases. Because PrP^(SC) is not readily degradedby the cell and accumulates to very high levels¹², even very smallamounts of contamination of suboellular fractions are sufficient to makedetection of subtle increases in ^(Ctm)PrP difficult. Thus, an alternatemethod is required to monitor the effect of accumulating PrP^(SC) on thetopology of newly synthesized PrP (see FIG. 11a).

[0144] In order to design such an experiment, we took advantage of threeobservations. First, a species barrier to PrP^(SC) conversion existsbetween mouse and Syrian hamster (Prusiner Cell (1990) 63:673-686;Pattison Res Vet Sci (1968) 9:408-410). Second, in contrast to thespecies barrier for PrP^(SC) formation, no species specific differencesin the synthesis, translocation or topology are observed between mousePrP (MoPrP) and SHaPrP (Hegde Science (1998) 279:827-834). And finally,monoclonal antibodies highly specific to SHaPrP (which do not crossreact with MoPrP) are available to distinguish between expression ofthese two PrP transgenes¹⁵. Thus, in such double transgenic animals wecan use hamster ^(Ctm)PrP formation as a ‘reporter’ during the course ofaccumulation of mouse PrP^(SC). For this experiment, the doubletransgenic mice which synthesize both MoPrP and SHaPrP are inoculatedwith mouse prions (of the RML strain). Then, at various intervals duringthe time course of accumulation of PrP^(SC) and development of disease,individual mice are sacrificed and examined for total PrP^(Sc)accumulation and for the presence of hamster ^(Ctm)PrP (see FIG. 4a).The principle is that following inoculation, only MoPrP will be asubstrate for prion replication and PrP^(SC) formation¹³. The effect ofthis PrP^(Sc) accumulation on the ability of cells to generate (or notgenerate) ^(Ctm)PrP can be assessed by examining SHaPrP.

[0145] Clinical disease was noted to develop in these animalsapproximately 9 weeks after inoculation (data not shown). We found thatPrP^(SC) accumulated in these mice during this 9 week time course, withthe earliest detectable times being approximately 5-6 weeks (FIG. 11b).As expected, the SHaPrP was not noted to have formed any PrP^(SC) byboth biochemical criteria in this study (FIG. 11b) and infectivitycriteria in prior studies¹³. Remarkably however, a significant increasein the amount of ^(Ctm)PrP was noted upon examination of the SHaPrP(FIG. 11c). Such an increase was not observed in a parallel set of micethat did not receive the inoculum (data not shown). These findings,coupled with the observation that ^(Ctm)PrP is capable of causingneurodegeneration in the absence of an transmissible forms of PrP (ref.9, FIGS. 8 and 10), suggest that PrP^(Sc) accumulation may cause diseaseby inducing the synthesis of ^(Ctm)PrP de novo.

[0146] The findings described herein suggest causal relationshipsbetween PrP^(SC) accumulation, the events of ^(Ctm)PrP formation andmetabolism, and the development of neurodegenerative disease. Threecomplementary and independent lines of evidence argue for thisconclusion. First, increasing the generation of ^(Ctm)PrP beyond acertain threshold (by modulating a combination of PrP mutation and levelof expression) results in neurodegeneration in the absence of PrP^(SC)formation (FIG. 8, FIG. 10 and ref. 9). Second, the amount ofaccumulated PrP^(SC) needed to cause neurodegenerative disease isinfluenced by the propensity of the host to generate ^(Ctm)PrP (FIG. 9).And third, the brain appears to contain increasing levels of ^(Ctm)PrPduring the course of accumulation of PrP^(SC) (FIG. 11). Taken together,the data are suggestive of three successive stages in the pathogenesisof prion diseases (FIG. 12).

[0147] Infectious prion diseases are proposed to work by initiating thesteps of Stage I, the accumulation of PrP^(SC). Genetic prion diseasescould in principle work at either Stage I or II. If the PrP mutation inquestion results in the spontaneous formation of PrP^(SC), Stage I wouldbe initiated, PrP^(SC) would replicate and accumulate, and subsequentlycause increased elevation of ^(Ctm)PrP (stage II). Such a mechanismseems plausible for the E200K mutation thought to cause certain geneticvariants of Creutzfeldt-Jakob disease¹⁶. Thus, PrP^(Sc) is seen in thesepatients' 17, and the disease is readily transmissible to experimentalanimals⁷. Alternatively, certain other PrP mutations could bypass stageI altogether by directly causing an increase in ^(Ctm)PrP generation.The A117V mutation resulting in human Gerstmann-Straussler-Scheinker¹⁸disease is likely to work by such a mechanism. This would explain whythis disease has not been transmissible⁶⁻⁸, and why PrP^(SC) has notbeen detected in these patients' brain tissue^(6, 9).

[0148] The final stage in prion disease pathogenesis includes themechanisms by which ^(Ctm)PrP, once generated, leads toneurodegenerative disease. The mechanism by which this occurs and theintracellular pathways that are involved remain entirely unclear.However, it does not appear to be the case that ^(Ctm)PrP is simplymisfolded, retained or accumulated in the ER, or eliciting an unfoldedprotein response. This is suggested by the observation that essentiallyall of the ^(Ctm)PrP has been trafficked beyond the ER⁹, the site of thepresently known quality control machinery for protein folding in thesecretory pathway^(19, 20). Additionally, disease can be elicited bytransgenes expressed at close to physiologic levels, as is the case withTg[SHaPrP(KH→II)_(M)] animals or human cases of GSS containing the A117Vmutation. Thus, a more selective means by which ^(Ctm)PrP inducesneurodegeneration is suggested by the available data.

[0149] The framework described in FIG. 12 suggests several new avenuesfor future studies. First, the regulated events in ^(Ctm)PrP biogenesisand trafficking remain to be elucidated. The reconstitution of the earlyevents of PrP translocation and topology determination in a cell-freesystem amenable to fractionation appears to be a promising avenue forthe identification of trans-acting factors regulating ^(Ctm)PrPsynthesis^(21, 22). Additionally, the availability of several mutantsinfluencing topology should facilitate studies aimed at defming laterevents in the trafficking of ^(Ctm)PrP. Second, insights gained fromstudying the metabolism of ^(Ctm)PrP will undoubtedly allow for a betterunderstanding of how these events are modulated in trans by PrP^(SC)accumulation. Such studies are likely to better delineate therelationship between the events of PrP^(SC) accumulation and ^(Ctm)PrPmediated neurodegeneration. Given that PrP^(SC) accumulation may impactupon several metabolic functions of the cell^(23, 24, 25), it isplausible that one or more of these influence ^(Ctm)PrP generation toelicit disease. The availability of PrP mutants that act at a stepbeyond PrP^(SC) accumulation to directly cause ^(Ctm)PrP mediatedneurodegeneration (ref. 9 and FIG. 8) should facilitate the dissectionof the downstream events in prion disease pathogenesis.

Example 3

[0150] Effect of Prolactin, Beta Lactamase, and Immunoglobulin G HeavyChain Signal Sequences on Protein Conformation

[0151] In general, a dichotomy has been accepted between a protein beingproperly folded versus misfolded (Ellgaard et al. (1999) Science 286;1882-1888). In other words, the possibility that proteins might havemore than one properly folded state and might even be able to select oneversus another folded state at different times or circumstances has notbeen considered. Because a linear polypeptide sequence folding indifferent ways could have substantially different shapes, physicalproperties and biological activities, this new level of regulationgreatly increases the information content of the genome and thepotential for regulation of gene expression available to the cell. Totest this hypothesis we first selected three unrelated, simple secretoryproteins, namely, prolactin (prl), beta lactamase (βlac), andImmunoglobulin G heavy chain (IgG), to determine whether the individualsignal sequences would be equally efficient at translocation of theirown passenger sequences across the ER membrane (see FIG. 13) as those ofdifferent nascent chains.

[0152] We truncated aliquots of these cDNAs at each of three uniquerestriction endonuclease recognition sites, so that when transcribed thetruncated cDNAs generated RNA transcripts encoding longer and longerC-terminally-extended lengths of these secretory proteins, and finallyexpressed these transcripts in cell-free translation systemssupplemented with microsomal membranes. As can be seen in FIG. 14,despite the fact that these three signal sequences are equally highlyefficient at translocating their own nascent chains, radically differentribosome-membrane junctions are established for translocation ofdifferent nascent chains, as determined by accessibility of the nascenttruncated chain to digestion with proteinase K (see cartoon to right ofeach panel of FIG. 14).

[0153] Initially upon targeting to the ER membrane, all three chainsremain accessible to protease (left hand panels of FIGS. 14A-C). Shortlythereafter, in the case of prolactin, a tight seal is established as thenascent chains is translocated directly to the ER lumen (see middlepanel of FIG. 14A). However, in the case of both beta lactamase and IgGheavy chain, the ribosome-membrane junction remains open at asubstantially later point (see middle panel of FIGS. 14B and C), andonly much later does it efficiently close (right hand panel of FIGS. 14Band C). This suggests that different proteins translocate to the ERlumen via strikingly different ribosome-membrane junctions. If, assuggested by the studies on prion protein (PrP) biogenesis, signalsequences are involved in regulation of protein folding, this couldoccur in part through establishing different environments in which thenascent chain starts to fold. When the ribosome-membrane junctionremains open, as in the middle panel of FIGS. 14B and 14C, the chainstarts to fold in the reducing environment of the cytosol; when theribosome-membrane junction is closed, as in the middle pane of FIG. 14Aand the right hand panels of FIGS. 14B and 14C, the unfolded or partlyfolded chain proceeds to the oxidizing environment of the ER lumen toinitiate or continue the process of folding funnel selection.

[0154] As shown above (Example 1) in the case of the biogenesis of prionprotein (PrP), the open and closed states of the ribosome membranejunction correlate to the synthesis of secretory versus Ctm forms ofPrP, identical polypeptide sequences that are folded differently and indifferent transmembrane topological forms. The data in FIG. 14 extendsthis correlation of the state of the ribosome-membrane junction with thefolding of the protein, to simple secretory proteins such as prolactinand immunoglobulin heavy chain.

[0155] If signal sequences play the hypothesized regulatory role inprotein folding, in part through the nature of the ribosome-membranejunction they establish, then swapping the signal sequence of twodifferent secretory proteins should have a dramatic consequence, in theform of three predictions:

[0156] First, swapping of the signal sequence should change theenvironment in which the nascent chain resides (e.g.as assessed eitherby a change in the ribosome-membrane junction as probed with proteinaseK, or by a change in the proteins with which the nascent chain isassociated as determined by chemical crosslinking). An example of achemical crosslink difference between nascent chains of prolactin behindthe prolactin signal sequence and prolactin behind the IgG signalsequence is shown in FIG. 15. Substantailly the same conclusion wasreached for PrP.

[0157] Second, the folding of the two proteins, directed down twodifferent folding funnels by virtue of interaction with differentaccessory proteins in different environments, is likely to be different.One way to score this would be by engineering in glycosylation sites asreporters of protein conformation and demonstrating that the same chainis glycosylated when it is behind one signal sequence but not another,even though both signal sequences are ultimately cleaved from theprotein. This is demonstrated in FIG. 16 for prolactin with anengineered glycosylation reporter, behind either its own signal sequence(Prl) or the signal sequence of immunoglobulin heavy chain (IgG/Prl),growth hormone (GH/Prl). As can be seen from the 5^(th) lane of eachpanel in FIG. 16A, the resulting mature prolactin chain made behinddifferent signal sequences is conformationally different as assessed bythe ability of oligosaccharyl transferase to add core N-linked sugars tothe nascent chains. FIG. 16B quantitates this difference. Expression oftruncations of these constructs reveal that the difference inglycosylation of one prolactin compared to another, is not a kineticphenomenon because prolonged incubation of the truncated and thereforenon-growing but nascent chains, did not result in any further degree ofglycosylation. FIG. 16C shows that this difference in conformation, asscored by glycosylation, is readily apparent from material secreted intothe medium of cos cells transfected with the corresponding constructsencoding prolactin behind the various signal sequences. Thus theconformation of prolactin is changed simply by virtue of its synthesisbehind a different signal sequence. That difference cannot be accountedfor by the known roles of the signal sequence, because all signalsequences utilized are equally competent for targeting and translocationof both their native substates (see FIG. 13) and that encoded by themodified prolactin coding region. Furthermore, all conformations ofprolactin synthesized in FIG. 16 are judged as properly folded by thequality control machinery, as assessed by their secretion into themedium.

[0158] A third prediction is that, in some cases of swapped signalsequences, the “mis-match” between the signal and the subsequentpassenger may be sufficiently severe that even fully targeted chains areincapable of finding a folding funnel compatible with translocation intothe ER lumen and may instead “fall back” into the cytosol, unable to betranslocated. Precisely this form of defect was observed for a PrPmutant that could only be expressed in the secretory form with intact ERmembranes, and whose expression in glycoprotein-depleted reconstitutedmicrosomal membranes resulted in an inability of the chain to betranslocated (Hegde et al, (1998) Mol Cel 2:85-91).

[0159] In FIG. 13 we demonstrate that behind the βlac and IgG signalsequences, prolactin displays a substantial translocation defect (FIG.13A), which which can be remedied by introduction of sequences from theregion of IgG subsequent to the signal sequence (FIGS. 13B and 13C).Furthermore the defect in mutant PrP translocation acrossglycoprotein-depleted reconstituted membranes can be corrected byswapping the PrP signal sequence for that of prolactin (data not shown),suggesting that the defect was neither intrinsic to the membranes northe chain, but rather, reflected an incompatibility of a particularsignal sequence-chain combination, consistent with the hypothesized roleof the signal sequence in folding funnel selection.

Example 4

[0160] From the above, it can be seen that, the signal sequence candetermine the final folded state of the protein, as demonstrated byaccessibility of a glycosylation site engineered into the protein behindsome signal sequences but not others. As the growing chain proceeds downdifferent folding funnels, depending on the nature of theribosome-membrane junction and protein-protein interactions between thenascent chain and the translocation machinery, different conformationsare achieved. As a result of those differences in conformation, evenwhile the chain is growing, the differently folded forms of a proteinare differentially accessible to modification enzymes. Hence, anengineered glycosylation site serving as a reporter of proteinconformation is glycosylated when engineered behind one signal sequencebut not another. Furthermore, in the extreme case where the nature ofthe ribosome-membrane junction is such as to allow substantial chainfolding to be initiated prior to translocation, additional informationin the authentic chain must emerge to close the ribosome-membranejunction. Without such additional information, the chain may fail totranslocate, as demonstrated for prolactin behind the IgG signalsequence.

[0161] Thus, a model secretory protein recapitulates the observationsmade previously for the prion protein (Example 1), indicating that thoseresults were not a special case. Indeed, what appears to be specialabout the prion protein is not the pathway of its biogenesis andfolding, but that the alternative folding pathways manifest astopological differences, making them far more easy to detect.

Example 5 Modulation of Protein Folding by Trans Acting Factors

[0162] As shown in Example 1, above, the time course of PrP^(Sc) accrualin transmissible prion disease is followed closely by increasedgeneration of ^(Ctm)PrP. Thus, the accumulation of PrP^(Sc) appears tomodulate in Trans the events involved in generating or metabolising^(Ctm)PrP. ^(Ctm)PrP, which has its C terminal domain translocated tothe ER lumen, triggers spontaneous neurodegeneration when overexpressed(Hegde, Science (1998) 279:827-834). As shown in Example 2, ^(Ctm)PrPappears to be induced just prior to onset of clinical signs, suggestingthat it initiates a final common pathway to neurodegeneration. An as yetunknown glycoportein of the ER membrane is implicated as a translocationaccessory factor (TrAF) that “protects” the normal brain from expressionof ^(Ctm)PrP by directing nascent PrP chains to the pathway leading to^(sec)PrP (Hegde Mol Cell (1998) 2:85-89).

[0163] The mechanism by which nascent PrP chains are allocated among thetopological forms is complex (Rutkowski, submitted). The PrP signalsequence itself may play a role by establishing at least two populationsof nascent chains: some with an open ribosome-membrane junction; otherswith a closed ribosome-membrane junction. Thus, the signal sequence candetermine the environment faced by the emerging N-terminal domain ofPrP. Only the chains exposed to the cytosolic environment have thepotential to become ^(Ctm)PrP. While necessary, an openribosome-membrane junction is not sufficient to make ^(Ctm)PrP, asadditional protein-protein interactions determine the final outcome.Mutations that prevent the transmembrane domain from directing ^(Ctm)PrPformation result in these chains being redirected to the cytosol, wherethey most likely are degraded.

[0164] The distinction between ^(sec)PrP and ^(Ctm)PrP usually are madeon topological grounds. However, these two polypeptides of identicalsequence also differ in their conformation, as determined by theirdifferential sensitivity to limited protease digestion in non-denaturingdetergent solutions. Thus, translocational regulation appears to be ameans of generating multiple forms of PrP that differ in bothconfomation and function. The machinery (i.e. a TrAF) that directsnascent PrP chains to make ^(sec)PrP rather than ^(Ctm)PrP, may itselfbe regulated, based on the ability of scrapie infection to increase theamount of ^(Ctm)PrP detectable in brain. Together, these observationslead to a new principle: a protein's conformation is determined not justby its primary amino acid sequence, but also by proteins such as TrAF,that influence which of two or more different functional conformationaloutcomes actually occur or predominate.

[0165] Complex secretory or integral membrane proteins therefore canhave many potential functional folded states. The different foldedstates become manifest by a combinatorial series of interactions betweenligands within the nascent chain and receptors at the translocon, in anarray of possible environments (e.g. cytosol, membrane, lumen). Therates of generation versus degradation of the various conformationsdetermine which, and how many, of the possible final outcomes for aparticular substrate are expressed at any given time. These processesare integrated by signaling pathways from the cell surface andelsewhere, which coordinate protein biogenesis with both the specificimmediate needs (Chapman Annu Rev Cell and Dev Biol (1998) 14:459-485)and the global program of the cell (Bonafacino Nature (1990)334:247-251)

[0166] This view necessitates that secretory and integral membranenascent chains proceed down more than one folding funnel (or takedifferent paths to different ends within a single, complex foldingfunnel). This is accomplished through the interaction of discretesequences in the nascent chain with TrAFs or the environments that TrAFsmake accessible. Different TrAFs, localized to different compartments(cytosol, membrane, ER lumen), that can act on different chains ordifferent subsets of folding states of a given chain also contributes tothe selection of one functional folded form versus another. This can bethought of as “third-order” complexity in protein folding. It differsfrom the concept of molecular chaperones in that each of the possiblestructural outcomes are functional—just different in some aspect offunction or regulation. Thus secretory and integral membrane proteinscould exist in multiple confomations, each with a distinctive functionalsignature.

[0167] Finally, the translocon and its components including TrAFs, thatdictate the choice among possible alternative folding funnels couldthemselves be subject to regulation by intracellular signaling,providing a “fourth order” of complexity to protein folding. FIG. 18summarizes a working model of translocational regulation and relates itto the four orders of protein folding.

[0168] In order to explore translocational regulation in trans we havedeveloped fractionated and reconstituted systems in which, as a resultof manipulation of either the cytosol or the membranes, theconformational mix of PrP can be altered. Since the findings with PrPhave been shown (Rutkowski and Lingappa) to be reproduced by studies inother unrelated proteins (Rutkowski and Lingappa; Lingappa, Buhman, andFarese) it is clear that these systems will be amenable to manipulationin order to achieve the goals of detecting and studying the conformersof any signal sequence containing gene product of interest.

[0169] Results

[0170] Previously, the evidence for trans-acting influences affectingtranslocation and conformation have been relatively indirect (Hegde etal. (1998) Mol Cell 2:85-91; Hegde et al. (1999) Nat. 402:822-826). Wehave now found two new lines of evidence that support this notion moredirectly. First we have observed that cytosolic extracts prepared frommouse erythroleukemia (MEL) cells before and after differenciation areable to affect translocation of PrP, shifting the distribution of formsin favor of Sec-PrP (see FIG. 19). Second, when microsomal membraneswere prepared from hamster brain at various times in early post-nataldevelopment, TrAF activity was found to vary, going from high in day 13embryonic brain, to a nadir at post-natal day 14, in a manner consistentwith the hypothesis that TrAF activity, which suppresses Ctm-PrP, isdecreased in selected brain regions to promote Ctm-PrP and triggerneuronal apoptosis as part of the program of early rodent braindevelopment (see FIG. 20).

[0171] In view of the results described above, we set about tofractionate the rabbit reticulocyte lysate in a manner that would beamenable to complementation with trans-acting factors from othersources. We found that a combination of ribosome pellet and DEAE eluatefraction were sufficient to restore full translational activity (seeFIGS. 21-23). The fractionation methodology is described above.

[0172] Similarly, we modified previously published protocols (Gorlichand Rapoport (1994) Cell 75:615-630; Hegde et al. (1998) Mol Cell2:85-91) to generate fractionated and reconstituted proteoliposomes thatwere functionally equivalent to previous preparations, but which hadadvantages in terms of cost, speed, and amenability to furtherfractionation. This protocol is described above.

[0173] It is evident from the above results that the subject inventionprovides a new platform and paradigm for detecting, magnifying,understanding and manipulating the participation of the translocationsystem in physiological processes and the effect of different conformersof any secretory or integral membrane protein, or other protein with asignal sequence for translocation across the ER membrane, on thephysiology of a host. By virtue of being able to control the folding ofa protein, where different conformers may lead to differentphysiological outcomes, the opportunity to investigate the effect of theconformers on cellular pathways, the identification of agents involvedwith the formation of the different conformers, treatment for adverseresults from a particular conformation and understanding of the cellularprocesses resulting from the formation of the different conformers, canbe achieved. New drugs can be developed and diseases can be consideredfrom different viewpoints than have been heretofore employed.

[0174] The above results also demonstrate that microsomal membranesprepared from different species with different genetic compositionprovide a powerful tool for the expressing normally minor conformers. Inthis case, sea urchin microsomal membranes were found to be highlytranslocation competent, but to generate only one conformer of PrP,Ctm-PrP, a form that is normally a minor species in the presence ofmammalian microsomal membranes. The potential advantage of this approachover the biochemical approach is that labile components are less likelyto have been inactivated by the procedure, and multiple components canbe distinguished by the presence of distinctive partial activities indifferent membranes. Also the preparation of membranes from tissues ofprimitive organisms may be more cost-effective, at least for an initialscreen, with positive results confirmed with the more expensive,biochemically pure reagents.

[0175] The novel protocol provided for the preparation of fractionatedand reconstituted proteoliposomes makes use of relatively inexpensivedetergents compatible with ion exchange chromatograpy, allowing forfurther fractionation of activities initially identified in the conA-depleted reconstitution, as well as activities whose initialidentification requires further fractionation of Con A flow-through. Afurther novel advantage oft he procedure provided here is that it makesthe initial screen for novel TrAFs prior to commitment of a majoreffort, quite non-labor intensive. This is especially important giventhe possibility that some TrAF activities may involve more than onecomponent and that, in some cases, one component in excess may be ableto partially compensate for lack of another component.

[0176] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims. All referencescited herein are incorporated herein by reference, as if set forth intheir entirety.

What is claimed is:
 1. A method for identifying conformers of a proteinmolecule having at least two different topographical forms, said methodcomprising: comparing the topography of a protein obtained by expressionof a chimeric gene having a DNA sequence encoding at least substantiallythe same amino acid sequence as an open reading frame encoding a nativeprotein, wherein said native protein is encoded by a gene having asignal sequence and said chimeric gene has a different signal sequence,wherein an alteration in topography of an expression product of saidchimeric gene as compared to an expression product of said native geneis indicative that said protein is a conformer of said native protein.2. The method according to claim 1, wherein said expression is in alysate.
 3. The method according to claim 2, wherein said lysate is afractionated lysate.
 4. The method according to claim 1, wherein saidexpression is in a system supplemented with a fractionated andreconstituted microsomal membrane
 5. The method according to claim 1,wherein said expression is in an intact cell.
 6. The method according toclaim 1, wherein the mature proteins resulting from expression of saidchimeric gene and said native gene are identical.
 7. A method forpreparing antibodies to a conformer other than the conformer of themajor normal wild-type protein, said method comprising: immunizing amammalian host with a protein obtained by expression of a chimeric genehaving a DNA sequence encoding at least substantially the same aminoacid sequence as an open reading frame encoding a native protein,wherein said native protein is encoded by a gene having a signalsequence and said chimeric gene has a different signal sequence so thata conformer is produced, under conditions whereby antibodies areproduced which bind to said conformer with an affinity different fromthat of said antibodies for said native protein.
 8. The method accordingto claim 7, further comprising: after immunizing, immortalizingB-lymphocytes from said mammalian host to provide immortalizedB-lymphocytes secreting antibodies; screening said antibodies for adifferent affinity for said different conformer than said conformer ofthe major normal wild-type protein to identify differentconformer-specific antibodies; and isolating said immortalizedB-lymphocytes secreting said different conformer-specific antibodies. 9.The method according to claim 8, wherein said mammalian host is a mouse,said B-lymphocytes are splenocytes and said immortalizing is withneoplastic cells to produce hybridomas.
 10. A method for detecting aconformational difference in a protein, said method comprising:contacting a non-denatured protein, obtained by expression of a chimericgene having a DNA sequence encoding at least substantially the sameamino acid sequence as an open reading frame encoding a native protein,wherein said native protein is encoded by a gene having a signalsequence and said chimeric gene has a different signal sequence so thata conformer is produced, with an agent that detectably modifiesaccessible side groups of amino acids common to said conformer and tosaid native protein to produce a conformer and a native proteincomprising modified side groups; and detecting differences in saidmodified side groups between said conformer and said native protein asindicative of a conformational difference between said conformer andsaid native protein.
 11. The method according to claim 10, wherein saidagent is a crosslinking agent or a chemical modification agent.
 12. Themethod according to claim 10, wherein said chemical modification agentis N-sulfo biotin or trinitrobenzenesulfonic acid.
 13. The methodaccording to claim 10, further comprising: evaluating reactivity of saidprotein of interest to a protease, wherein altered reactivity isindicative of a conformational difference in said protein as compared tosaid native protein.
 14. A method for detecting a conformationaldifference in a protein of interest, said method comprising: analyzing anon-denatured protein, obtained by expression of a chimeric gene havinga DNA sequence encoding at least substantially the same amino acidsequence as an open reading frame encoding a native protein, whereinsaid native protein is encoded by a gene having a signal sequence andsaid chimeric gene has a different signal sequence so that a conformeris produced, by high pressure liquid chromatography, wherein analteration in the chromatographic profile of said protein of interest ascompared to a native protein is indicative of a conformationaldifference.
 15. The method according to claim 14, wherein said highpressure liquid chromatography is performed using hydrophobic adsorptioncolumns.
 16. A method for identifying the presence of a conformer in asample, wherein at least two different conformers exist, from a proteinfor which conformers have not been identified, wherein the conformerdifferent from the major wild-type conformer is produced by expressionin a cellular host, and the said assay comprising: combining said samplewith an entity having an altered binding affinity for said differentconformer as compared to said wild-type conformer; and detecting thebinding of said entity as an indication of the presence of saiddifferent conformer.
 17. The method according to claim 16, wherein saidentity is an antibody.
 18. A method according to claim 16, wherein saidentity is a labeled molecule of less than 5 kDal.
 19. A DNA constructcomprising a DNA sequence for a PrP protein joined to a non-nativesignal sequence.
 20. A DNA construct according to claim 19, wherein saidsignal sequence results in the formation of Ctm-PrP.
 21. A non-humanmammal comprising a DNA construct according to claim
 19. 22. A method ofevaluating individual responses to environmental changes associated witha disease state, said method comprising: subjecting lymphocytes fromsaid individual to said environmental change; and determining the changein expression of Ctm-PrP in said lymphocytes, as an indication ofindividual responses to environmental change.
 23. A method for screeningindividuals for susceptibility to a disease associated with Ctm-PrP,said method comprising: assaying for the level of Ctm-PrP in a cell,tissue or organ affected by said disease or bodily fluid associated witha cell, tissue or organ affected by said disease of said individual, asrelated to the level in control cells, tissues, organs or bodily fluids;or assaying for a change in level of Ctm-PrP in response to an externalagent in disease related cells as compared to the level of Ctm-PrP inresponse to an external agent by normal cells as an indication of thesusceptibility to a disease associated with Ctm-PrP.
 24. The methodaccording to claim 23, wherein said disease is a neurodegenerativedisease or a myopathy.
 25. The method according to claim 23, whereinsaid tissue is a brain tissue or a muscle tissue, said cell is alymphocyte and said bodily fluid is cerebrospinal fluid.
 26. A methodfor screening compounds for their effect on the relative amounts ofdifferent conformers of a protein, said method comprising: combiningcells with said compound for sufficient time for formation of saidprotein and any different conformers thereof; and assaying for alteredamounts of said different conformers as compared to amounts of saiddifferent conformers in the absence of said compound.
 27. A method forscreening compounds for their effect on the relative amounts ofdifferent conformers of a protein produced in a mammal, said methodcomprising: providing a mammal with said compound; and determining thechange in relative amounts of conformers of said protein as compared tothe relative amount of said conformers in the absence of said compoundin a bodily fluid or tissue of said mammal.
 28. The method according toclaim 27, wherein said protein is PrP.
 29. A method for determining theeffect of an agent on the relative amounts of the conformers of anendogenous protein, said method comprising: contacting a chimeric hostwith said agent, wherein said chimeric host is characterized by having aforeign gene encoding said protein from a different species or otherthan an endogenous gene processed by the same translocational mechanismas said gene encoding said protein; and determining the effect of saidagent on said foreign gene as a surrogate for said endogenous gene. 30.One or more proteins having an altered conformation, said one or moreproteins produced by the method of: obtaining expression in afractionated lysate of a chimeric gene having a DNA sequence encoding atleast substantially the same amino acid sequence as an open readingframe encoding a native protein, wherein said native protein is encodedby a gene having a signal sequence and said chimeric gene has adifferent signal sequence, wherein said protein is other than Ctm-PrP,whereby one or more proteins having an altered conformation areproduced.
 31. One or more proteins having an altered conformation, saidone or more proteins produced by the method of: obtaining expressionusing fractionated and reconstituted membranes of a chimeric gene havinga DNA sequence encoding at least substantially the same amino acidsequence as an open reading frame encoding a native protein, whereinsaid native protein is encoded by a gene having a signal sequence andsaid chimeric gene has a different signal sequence, wherein said one ormore proteins is other than Ctm-PrP, whereby one or more proteins havingan altered conformation are produced.
 32. A method for alteringconformation of a protein, said method comprising: obtaining expressionof a chimeric gene having a DNA sequence encoding at least substantiallythe same amino acid sequence as an open reading frame encoding a nativeprotein, wherein said native protein is encoded by a gene having asignal sequence and said chimeric gene has a different signal sequence,whereby at least one protein having an altered conformation as comparedto said native protein is obtained.
 33. The method according to claim32, wherein said expression is in whole or fractionated lysatessupplemented with whole or fractionated and reconstituted membranes fromone or more organisms that lack or have altered genes that encode one ormore protein with which signal sequences, nascent chains ortranslocational machinery associated with expression of said nativeprotein interact.
 34. The method according to claim 32, wherein saidorganisms occur in nature.
 35. The method according to claim 32, whereinsaid genes that said organisms lack encode trans-acting factors.
 36. Themethod according to claim 32, wherein said organisms are sea urchinembryos.
 37. One or more mutant or chimeric proteins having an alteredconformation, wherein said one or more proteins are produced by themethod according to claim 31, wherein said one or more proteins areother than Ctm-PrP.
 38. The method according to claim 32, wherein saiddifferent signal sequence is a regulatable signal sequence.
 39. Themethod according to claim 38, where said regulatable signal sequence isregulated by one or more trans-acting factors.
 40. A method forclassifying a signal sequence, said method comprising: obtainingexpression of multiple chimeric genes each having a DNA sequenceencoding at least substantially the same amino acid sequence whereineach chimeric gene has a different signal sequence, under at least twoconditions whereby one or more proteins having an altered conformationare produced, wherein said conditions include a. a fractionated lysate;b. fractionated and reconstituted membranes; and c. a fractionatedlysate supplemented with whole or reconstituted membranes from amammalian organism that lacks or has altered genes or supplies a missinggene for one or more proteins with which signal sequences, nascentchains or translocational machinery associated with expression of saidnative protein interact; d. a fractionated lysate supplemented withwhole or reconstituted membranes from a non-mammalian organism thatlacks or has altered genes or supplies a missing gene for one or moreproteins with which signal sequences, nascent chains or translocationalmachinery associated with expression of said native protein interact;and e. a cytosol mix; evaluating expression products obtained from saidmultiple chimeric genes under said at least two conditions as indicativeof a classification of each said different signal sequence.
 41. Themethod according to claim 40, wherein said DNA sequence encodes or PrPor prolactin comprising engineered glycosylation acceptor sites asreporters of change in conformation of said prolactin.
 42. The methodaccording to claim 40, wherein said expression products are evaluated bya method selected from the group consisting of reactivity to proteasetreatment, high pressure liquid chromatography, modification of sidegroups.
 43. A method for identifying a change in quantitativedistribution of conformers of a protein molecule having at least twodifferent forms, said method comprising: comparing the distribution ofconformations of said protein in multiple biopsy samples obtained froman animal; and determining the amount of each conformation expressed insaid multiple biopsy samples as indicative of a change in quantitativedistribution of said conformers.
 44. The method according to claim 43,wherein said multiple biopsy samples are taken from different tissues insaid animal.
 45. The method according to claim 43, wherein said multiplebiopsy samples are taken at different time intervals.
 46. An isolatedcell or a recombinant non-human cell comprising: one or more conformersof a disease associated protein, wherein said conformer of said diseaseassociated protein is other than Ctm-PrP.
 47. The isolated cell orrecombinant non-human cell according to claim 46, wherein said diseaseis a neurodegenerative disease or a myopathy.
 48. An isolated orpurified conformer of a disease associated protein, wherein saidconformer of said disease associated protein is other than Ctm-PrP. 49.An isolated or purified conformer of a disease associated protein,wherein said conformer is identified according to the method of claim 1.50. A chimeric gene comprising: an open reading frame encoding a diseaseassociated protein and a signal sequence other than the signal sequencenative to said protein.
 51. The chimeric gene according to claim 50,wherein said disease associated protein is a prion protein.
 52. Anisolated cell or a recombinant non-human cell comprising: a nascentchain of a disease associated protein bound to a trans-acting factor.53. An isolated cell or a recombinant non-human cell comprising: a geneencoding a disease associated protein wherein a signal sequence of saidgene is bound to a trans-acting factor.
 54. The isolated cell ofrecombinant non-human cell according to claim 52 or claim 53, whereinsaid trans-acting factor is capable of altering the conformation of saiddisease associated protein.
 55. The isolated cell of recombinantnon-human cell according to claim 52 or claim 53, wherein said diseaseassociated protein is a prion protein.